Technology Poster Single-Cell PB 2020 DPI 041420 VIEW · AAAAA mRNA UMI Barcode Read1 TTT mRNA...

transcript

Key Yellow highlights indicate the target of the protocol

Single-Cell For all you seq...RNA Low-Level Detection

DP-SeqDesigned Primer-based RNA-se-quencing strategy (DP-seq)

DNAcDNA

AA(A)n

De�ne set of heptamer primers

Poly(A) selection First strand cDNA synthesis

Hybridize primers PCR

AA(A)n TT(T)n

No secondary structure

Unique sequenceAA(A)n

TT(T)n

Digital RNAHiRes-SeqFREQ-SeqRNAtag-Seq

cDNAcDNA1

Amplify SequenceUnique molecular barcodes are added after cDNA synthesis for quantitative allele frequency detection. High-resolution RNA-seq to assess noncoded base substitutions in mRNA (HiRes-Seq)

Adapters with unique barcodes

Align sequences and determine actual ratio based on barcodes

Some fragments amplify preferentially

True RNA abundance

mRNASmart-SeqNanoCAGE AAAAAAA

mRNA fragment

AAAAAAA

Second strand synthesis

AAAAAAAT T T T T T T

T T T T T T T

Adapter

AdapterSwitch mechanism at the 5’ end of RNA templates (Smart)

PCR ampli�cation PurifyFirst-strand synthesis with MMLV reverse transcriptase

CCCCCC

mRNAUMI Method AAAAAAA

mRNA fragment

AAAAAAA

First strand synthesis Second strand synthesis

AAAAAAAT T T T T T T

True variant

Random errorDNA

T T T T T T T

Degenerate molecular tag (N10)

Unique molecular identi�ers (UMIs) uniquely identify copies derived from each molecule

PCR ampli�cation Align fragments from every unique molecular tag

CCC CCC

mRNASmart-Seq2 AAAAAAA

mRNA fragment

AAAAAA

cDNA synthesis Tagmentation

AAAAAA AAAAAAT T T T T T T T T T T TAdapter

Switch mechanism at the 5’ end of RNA templates (Smart)

PCRFirst-strand synthesis with MMLV reverse transcriptase

CCCCCC GGGTem-plate-switch-ing oligo

Locked nucleic acid (LNA)

CCCGGG

Enrichment-ready fragment

Index 1Index 2

Gap repair, enrich-ment PCR and PCR puri�cation

Single-cell tagged reverse transcription (STRT)

AA(A)n

AA(A)nCell 1

Cell 2

Cell 3

TT(T)n

AA(A)n

AA(A)nTT(T)n

TT(T)n

cDNA synthesis

Add 3 to 6 cytosines

TT(T)n

CCCGGG

Template-switch-ing primer

Introduce unique index

Add oligo(dT) primer Pool Single-primer PCR and purify

Separate cell sequences based on unique indices

Cell 3

Cell 2

Cell 1TT(T)n

Unique index

5’ adapter

CEL-SeqAA(A)n

AA(A)n

AA(A)nCell 1

Cell 2

Cell 3T7promoter

Unique index

5’ adapter

TT(T)n

AA(A)n

AA(A)nTT(T)n

TT(T)n

Second strand RNA synthesis

Fragment, add adapters and reverse-transcribe

Separate cell sequences based on unique indices

PoolCell 3

Cell 2

Cell 1

Cell expression by linear ampli�ca-tion and sequencing (CEL-Seq)

cDNA synthesis TagmentationPCRFirst strand synthesis

AAAAAAT T T T T T

AdapterCCC AAAAAA

T T T T T TCCCGGG

CCCGGG

Index 1Index 2

Gap repair and PCR

Single-nuclei RNA sequencing (snRNA-seq)

snRNA-Seq AA(A)n

Singlecell polyA RNA

Cell suspension

Lyse and centrifuge

Sort nuclei

Supernatant

NucleiNucleus

cDNA synthesis TagmentationPCRFirst-strand synthesis

AAAAAAT T T T T T

AdapterCCC

AAAAAAT T T T T TCCC

GGGCCCGGG

Index 1Index 2

Gap repair and PCR

Fixed and recovered intact single-cell RNA (FRISCR)

FRISCR AA(A)n

Fixedsingle cell polyA RNA

Cell suspension

Fix Sort single cells

Isolate RNA

Lyse cells and reverse crosslink

AAAAAA

Quartz-SeqWhole-transcript ampli�-cation for single cells(Quartz-Seq)

AA(A)n AAAAA AAAAAT T T T T

TTTTT T7 PCR

Add poly(A) primer with T7 promoter and PCR target

AAAAAT T T T T

Reverse transcriptionand primer digestion

T7 PCR T7 PCR

Poly A addition and oligo dT primer with PCR target

Generate second strand

Add blocking primer

Enrich with suppres-sion PCR

T T T T TPCR

T T T T T T7 PCRAAAAAT T T T TPCR AAAAA

TTTTT T7 PCRAAAAA

Blocking primer with LNA

MARS-SeqMassively parallel RNA single-cell sequencing framework (MARS-Seq)

AA(A)n AAAAA

TTTTTT7UMI

Add poly(A) primer with partial T7 promoter and UMI

Second strand synthesis

RNA fragmentation RNA to ssDNA ligationDNaseI Reverse transcription

PCR and puri�cation cDNA

AAAAAT T T T T

partial rd1rev

scRNA-seqSingle-cell mRNA sequencing (scRNA-seq)

AA(A)n AAAAA

Add polyT primer

Reverse-transcribePoly(A)-tailed mRNA

Reverse transcription and primer digestion with ExoSAP-IT

PCR ampli�cation Shear DNA

AAAAAT T T T T

T T T T T AAAAAT T T T T

T T T T TAAAAA

Corrected sequenceIdentify low abundance RNA viruses with circular sequencing (CirSeq)

CirSeqWhole-genome RNA

AA(A)n

Circularize RNA with kinase and RNA ligase 1

Random primers

Circular RNA template

Repeat 1Repeat 2

Repeat 3

Repeat 1Repeat 2Repeat 3

Mutation

Transcriptome in vivo analysis (TIVA)

TIVA Whole-genome RNAAA(A)n

Capture on Streptavidin coated magnetic beads

mRNA from single cell

AAAAAAA AAAAAAA

UUUUUUUUUUUUUUUUUUCy3

Cy5 PLCPP

Biotin

CPP Cell-penetrating peptide

Disul�de bondS SPhotocleavable linkerPL

UUUUUUUUUUUUUUUUUUCy3 UUUUUUUUUUUUUUUUUUCy3

CPP SAAAAAAA AAAAAAA

Cy5 PLPL

AAAAAAA AAAAAAA

Cy5 PL PLS AAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA

Load into cells

CPP peptide released

Photoactivate Anneal to mRNA

Gene expression cytometry (CytoSeq)

CytoSeqBarcoded mRNA from single cells

AA(A)n

Singlecell

Cell suspension

Each bead with unique oligos

Load cells and beads into microwells

Cell lysis, mRNAshybridize on bead

Pool all beads from microwells

cDNA synthesis and ampli�cation

Sequence

UniversalCell labelMolecular indexOligo(dT)

Analyze mRNA transcripts from individual cells in droplets (Drop-seq)

Drop-Seq

Barcoded mRNA from single cells

AA(A)n

Singlecell

Cell suspension

Each bead with unique oligos

Load cells and beads into droplets

Cell lysis, mRNAshybridize on bead

Pool all beads from droplets

Sequence

TCRα mRNA

TCRβ mRNA

Oil emulsion

Identify T-cell Receptor (TCR) alpha–beta chain pairing in single cells

Reverse transcription

Ampli�cation Overlap extension Blocker primers

Nested PCR ampli�cation

TCR Chain Paring

AA(A)n

TCRα TCRβ TCRα TCRβTCRα TCRβ

DNAPCR suppression of non-fused molecules

CDR3α CDR3β

Whole-genome RNA

Peptide nucleic acid-assisted identi�cation of RNA binding protein (PAIR)

Capture on magnetic beads

Visualize protein on SDS-PAGE

CPP Cell-penetrating peptide

Disul�de bondS SPhoto-activatible compoundBpa

Binding site for RNA-bind-ing protein

Create peptide nucleic acid analogs (PNAs)

AA(A)n AA(A)n

PNACPP S S

PNABpa

CPP peptide released

BpaCPP S

PNABpaS

AA(A)n AA(A)nS S Bpa

PhotoactivateLoad into cells

AA(A)n

Cell labeling via photobleach-ing (CLaP)

CLaPBarcoded mRNA from single cells

AA(A)n

Singlecell

Con�uent cells in culture

Biotin-4-�uo-rescein (B4F)

Photobleach and crosslink with 473 nm laser

Cy5-streptavidin labeling

Tagged cells isolated, reverse-transcribed and sequenced

TCR-LA-MC PCR TCR ligation-anchored-mag-

netically captured PCR (TCR-LA-MC PCR)

Constant (C) gene of the TCR chains

(C) gene cDNA synthesis

RNA digestion

ssDNA linker ligation

Index 1Index 2

PCR 2 with nested primers

Add sequencing adapters

DNA ready for sequencing

CCCCCCCCCCCCCCCCCCCC

MALBAC primers

RNAUMI

PolyA RNASingle cell

Non-polyA RNA

RNA digestion and dC tailing

G-enriched MALBAC primers with UMIs

GGGGGGGGGGCCCCC

Cell lysis

Repeat 9x

Multiple annealing and dC-tailing-based quantita-tive scRNA-Seq (MATQ-Seq)

MALBAC primers initiate quasilinear ampli�cation to reduce ampli�cation of PCR bias

Primer hybridization at low temperatures increases RT e�ciency and non-polyA transcript detection

Addition of dC tail to 1st cDNA strands increases e�ciency of 2nd strand synthesis

UMIs drastically reduce exponential PCR ampli�cation bias

Library prep and sequence

AA(A)nMATQ-seq

Random displacement ampli�cation sequencing (RamDA-Seq)

Strand displacement ampli�cation assisted by gp32

2nd cDNA strand synthesis with NSR primers and Klenow Fragment

Tn5 transposase-mediated library prep

Random nicks on cDNA by DNase I

Not-so-random (NSR) primers prevents binding with rRNA

1st cDNA strand synthesis with RNase H minus RTase

AA(A)n RNA

cDNA3’5’

DNase Igp32

Non-polyA RNA

PolyA RNANSR primerCell

lysisSingle cell

NSR primerRamDA-seq

AA(A)n

Singlecell

Nuclei

Microchannel containing nuclei

Microchannel containing cytoplasm

Cytoplasm

Lysis and ITP extraction

Load into micro�uidics system

ITP acceleration isolates cytRNA

nucRNA-Seq by SMART-Seq2

Custom micro�uidics with isotachophoresis (ITP) bu�er chemistry

cytRNA-Seq by SMART-Seq2

Nuclear RNA

Cytoplasmic RNASequencing-ready

Sequencing-ready cDNA

SIngle-cell integrated nucRNA & cytRNA sequencing (SINC-Seq)

Correlation of gene expression between nuclei and cytoplasm from a single-cell

Nuclei is prevented from mixing with cytoplasmic material by hydrodynamic traps. Voltage manipulation physically separates cytoplasmic material following cell lysis

SINC-seq

Virus-infected single cell

Host RNA

Viral RNA

Virus-inclusive single-cell RNA-Sequencing (viscRNA-Seq)

Virus-infected cells are sorted by FACS into 384 well-plates and lysed

Host mRNA are captured using polyT oligos. Viral DNA captured by virus-speci�c oligos

5’-blocked template-switching oligos are used to reduce formation of concatemeric artifacts

AA(A)n Host RNA cDNA

Sequencing -ready cDNA

Well containing single-cell

Viral RNAAA(A)n

Infected cellsFACS sort into well plates Lyse

RT and template-switching Amplify and

library prepCapture RNA

Modi�ed SMART-Seq2

viscRNA-seq

AAAAAT T T T T AAAAA T T T T T AAAAA T T T T T

T T T T TPolyA RNA

AA(A)n

Single-cell in PCR well plate

Cell barcode

mRNAPCR primer

PolyA tail synthesized by TdT enzyme

PCR primer with polyT tail

2nd strand synthesis

cDNA library ready for sequencing

Single cell Lysis & RT

Pool & purify

Amplify & custom library prep

High-throughput single-cell RNA-Seq method that e�ectively uses limited sequence reads (Quartz-Seq2)

Single cells are sorted into PCR well plates containing lysis bu�er and RT primers (cell barcode, UMI, and oligo dT)

WTA using terminal deoxynucleotidyl transfer-ase (TdT) provides 3.6 fold increase in polyA tagging to UMI conversion e�ciency

Custom library prep attaches sequencing adapters and pool barcodes to enable mixing of di�erent sets of cell barcode-labeled cDNA

Quartz-seq2

Fixed cells

Barcode 1 Barcode 2 Barcode 3 Barcode 4

RT with 1st barcode set

Ligate 2nd barcode set

Ligate 3rd barcode set

Amplify with 4th barcode set

PCR of cDNA using 4th well-speci�c barcode along with sequenc-ing adaptors

Fixed and permeabilized cells are reverse-tran-scribed using microwell- speci�c unique barcodes (yellow for this well)

Each cell can be distin-guished by unique barcode sequences attached to cDNA

Barcode 3 contains UMIs and conjugat-ed biotin molecule

Ready for library prep

Random split

Random split Lysis and

Random split Pool PoolBiotin

Single cell

Split-pool ligation-based transcriptome sequencing (SPLiT-Seq)

PolyA RNAAA(A)n

SPLiT-seq

2nd index tagging and sequencing adapters

Well-speci�c transposomes

i5 adapter with cell index

i7 adapter with cell index

PoolIndex 1

Index 2

UMISingle cell

Single-cell combinatorial indexed RNA sequencing (sci-RNA-Seq)

Fixed cells or nuclei are randomly sorted and reverse transcribed using polyT adapters with UMIs and well-speci�c index

cDNA strands are tagged with another set of well-speci�c barcodes that includes i7 and i5 sequencing adapters

Ready for sequencing

RT with 1st barcode set

Fixed cellsRandom split

Random splitPool 2nd strand

synthesisPolyA RNAAA(A)n

sci-RNA-Seq

Each bead has unique cell-identifying oligo sequence

Load nuclei and beads into droplets

Break droplets & pool beads

OligobeadsDroplets

Droplets (enlarged)

PCR handleCell barcodeUMIOligo(dT)

AAAAAAAA

Single nuclei

Load nuclei into droplet micro�uidic system

Single nuclei droplet-based sequencing (snDrop-Seq)

PolyA RNA

Nuclei suspension

Single nuclei

AA(A)n

Nuclei lysissnDrop-Seq

Massively parallel single-nucleus RNA sequencing with droplet technology (DroNc-seq)

AA(A)n

Cell suspension

Barcoded mRNA from single nuclei

Pool all beads from droplets

Sequence

LysePolyA RNA

Oligobeads

Single nuclei Isolate

nucleiAAAA

non-polyA RNA

DroNC-Seq

DNA Low-Level Detection

MALBACGenome

Hybridize primers PCR

27-bp common sequence8 random nucleotides

Partial amplicons

Template

Denature

Hybridize primers Synthesis

Multiple annealing and looping-based ampli�cation cycles (MALBAC)

Cycles of quasilinear ampli�cation

Looped full amplicons

Bst DNA polymerase

Genomic DNA

GenesmMIP

Copy target sequence Exonuclease Corrected sequence

Align fragments from every unique molecular tag

Sample indexRead1

True variant

Random errorSingle Molecule Molecular Inversion Probes (smMIPs) for detecting low frequency targets

PCR ampli�cation

Degenerate molecular tag

Targeted STR

Short tandem repeat (STR)MIPSTRCopy target STR Amplify and sequenceTargeted capture of STR

loci by smMIPs (MIPSTR)

Degenerate molecular tag Strain I

Strain IIStrain I

Strain INatural variation between individuals Somatic variation

within an individual

Nuc-SeqSNES

Cell 1

Cell 2

Cell 3

Cell sorting from G2/M distribution

Lyse cell NucleusSingle G2/M nucleus sequencing of cells in S phase (nuc-seq)Single nucleus exome sequencing (SNES)

Single cell genome

phi29 Limited ampli�cation S1 nucleaseSynthesis DNA

GenomeMDAIMS-MDAMIDAS

Primer hybridization

Nascent replication fork

phi29 phi29 S1 nuclease Ampli�ed DNA

3’ blocked random hexamer primers

Synthesis SynthesisMultiple displacement ampli�cation (MDA) Immunomagnetic separation for targeted bacterial enrichment for MDA (IMS-MDA) Microwell displacement ampli�cation system (MIDAS)

Cell lysis

Size select

Break emulsion, RT, & template switching

Each antibody tagged with unique barcode sequence to allow sequence-based antibody ID

Antibodies bind to cell-surface proteins on cells

Single-cells and beads are loaded into droplets

mRNA and oligos from antibodies both have polyA tails, here they anneal to oligobead at its polyT end

dscDNA from cellular mRNA are considerably longer than dscDNA from antibody-oligo complex sequences

Cell-speci�c transcripts are identi�ed by barcode sequences and couple gene expression data with cell-surface protein marker data

Cellular indexing of transcriptomes & epitopes by sequenc-ing (CITE-Seq)

AntibodyAntibody barcode

Single cell

CellsLoad into droplets

PolyA RNA

Surface protein

AA(A)n

AAAAAAAA

AA(A)nS S

OligobeadsAntibody-oligo complex

dscDNA

From cellular mRNA

From antibodies

non-polyA RNA

CITE-Seq

Single-cells and beads are loaded into droplets

mRNA and oligos from antibodies both have polyA tails, here they anneal to oligobead at its polyT end

dscDNA from cellular mRNA are considerably longer than dscDNA from antibody-oligo complex sequences. sgRNAs have their own PCR primers

Transcripts can be identi�ed to cellular level through its cell-barcodes, connecting multiple modalities within a single experiment

Single cell PolyA RNA

Protein markers & sample tagging

AA(A)n

Sample A

Sample B

Sample C

Oligobeads

AntibodyAntibody barcode

AA(A)nS S

Antibody-derived tags

Antibody

AA(A)nSample A barcode

Antibody

AA(A)nSample B barcode

Antibody

AA(A)nSample C barcode

Cell lysis

AAAAAAAA

non-polyA RNA

Load into

drop-lets

Size select

From cellular mRNA

From antibodies

From sgRNAs

Break emulsion, RT, & template switching

dscDNA

Sample tagging to ubiquitous protein markers

Epitope-speci�c tagging Droplet microfuildics

Expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-Seq)

Hashtag oligos (HTOs) target speci�c proteins with barcodes to provide sequence-based cellular identi�cation of sample origin.

Antibody-derived tags mark cellular antigens of interest

ECCITE-Seq

AA(A)n

AAAAAAAA

PolyA RNAgRNA

CRISPR/Cas9Single cell

Cell suspension gRNA

construct

Drop-Seq

Lentiviral transduction & enrichment

Transcriptome

Puromycin selection enriches successful gRNA-transduced cells

scRNA-seq directly link transcriptome pro�le with corresponding guide RNA

cDNA libraryBreak droplets & library prep

Pooled CRISPR screens with single-cell transcrip-tome resolution (CROP-Seq)

Custom lentiviral construct contains gRNA in polyA mRNA transcripts so they can be detected through scRNA-Seq

CROP-Seq

Oligobeads

AA(A)n

AAAAAAAAAAAAPolyA RNAgRNA

CRISPR/Cas9Single cell

Cell suspension

gRNA constructViral

infection & enrichment

Transcriptome

Puromycin selection enriches successful gRNA induced cells

scRNA-seq directly link transcriptome pro�le with corresponding guide RNA

cDNA libraryBreak droplets & library prep

Mosaic single-cell analysis by indexed CRISPR sequencing (Mosaic-Seq)

Cellular enhancer activity perturbed by viral transduction of gRNAs, followed by antibiotic selection

Mosaic-Seq

Drop-Seq

Oligobeads

Minimize arti�cially induced transcrip-tional perturbations followed by scRNA-Seq (Act-Seq)

Actinomycin D (ActD) inhibits dissocia-tion-induced gene expression common in conventional dissociation methods

Load nuclei and beads into droplets

Break droplets & pool beads

OligobeadsDroplets

Droplets (enlarged)

AAAAAAAA

Load into droplet micro�uidic system

Single cell

Cell lysispolyA RNA

Singlecell

Frozen tissue slice Drop-SeqCell dissociation followed by Actino-mycin D treatment

AA(A)n

Act-Seq

PDMS subnanoliter array Oligobeads

Cell suspension

Simple, portable platform for massively parallel scRNA-Seq (Seq-Well)

Individual cells trapped in PDMS nanoarray wells

All oligos in the same bead contain identical cellular barcodes but unique UMIs

PolyA RNA captured by oligobeads

Wells are sealed with a semi-permeable membrane

Barcoded cDNA library

Cell lysis

Captured mRNA prepared for sequencing

polyA RNA

Singlecell

AA(A)n

Seq-Well

Agarose microwell array Oligobeads

Cell suspension

Single-cell

mRNAAdaptors

Transcriptomic pro�les for thousands of single-cells (Microwell-Seq)

Individual cells trapped in agarose microarray wells

All oligos in the same bead contain identical cellular barcodes but unique UMIs

PolyA RNA captured by oligobeads

Barcoded cDNA library

Cell lysis

Captured mRNA enriched using SMART-Seq2

polyA RNA

Singlecell

AA(A)n

UMICell labelMolecular indexOligo(dT)

Microwell-seq

Nanogrid single-nuclei RNA sequencing (Nanogrid SNRS)

Nanowells on the nanogrid are lined with custom oligos for polyA RNA capture and cDNA synthesis

Stained nuclei or cells are loaded into nanowells by nanodispensor

Viable vs. nonviable wells sorted by using �uorescence imaging

Fluorescent microscopy imaging

polyA RNASingle cell or nuclei

AA(A)n

Nanogrid

Oligo-lined nanowells

None/dead in well

More than 1 per well

1 nucleus/cell per wellDAPI-stained

nuclei

DispenseSelect viable wells & lyse

RT, template switching, and one-sided P7 tagmentation

Barcoded cDNA library �anked by sequencing adapters

cDNA library generation

P5 adapter

Well barcode

Oligo(dT)

cDNANanogridSNRS

PoolscRNA-Seq work�ow

Singlecell

Cell cultures with di�erent treatments

LMO-labeled cellsLipid-modi�ed oligonucleotide (LMO) with barcode

LMO sample barcodes are identical within a treatment group but unique among di�erent treatments

Rapid, modular, and universal scRNA-Seq sample multiplexing strategy using lipid-targeted indices (MULTI-Seq)

LMOs remain adhered to surface membranes until cell lysis during scRNA-Seq

Sequence analysis of LMO sample barcodes can identify treatment group, while cellular barcodes can identify individual cells

Sample barcode

5’ PCR handle

mRNAAA(A)n AAAA AAAAMULTI-Seq

Hybridize primers

Nascent replication fork

phi29 phi29 S1 nuclease Sequence and feed into SCcaller

3’ blocked random hexamer primers

Synthesis Synthesis

Genome

Perform in low temperatures

Single cell

Cell suspension Isolate

single-cells & lyse

Single-cell multiple displacement ampli�cation (SCMDA) with single-cell variant caller (SCcaller)

Linear ampli�cation via transposon insertion (LIANTI)

Single-cells lysed and gDNA tagmented by the LIANTI transposome

T7 RNA polymerase binds to T7 promoter site on tagmented gDNA

Linear ampli�cation of gDNA signi�cantly reduces ampli�ca-tion bias and replication errors

RNase digestion and 2nd strand cDNA synthesis and UMI barcoding

Genome

gDNA RNA cDNAUMIcDNA/RNA hybrid

LIANTI Transposome

LyseTransposase

T7 RNA Polymerase

Transposase bind site

T7 Promoter

Single cellLIANTI

Pool Pool

Transposase-based tagging

Random sorting

Isolate nuclei and nucleosome depletion

TransposomeDNA

DNASingle cell

PCR-based tagging

Random sorting

Single-cell combinatorial indexed DNA sequencing (sci-DNA-Seq)

Nucleosome depletion via SDS cross-linking

Randomly sorted nuclei are tagmented with well-speci�c unique indexed adapters

15-25 cells are randomly sorted into wells for 2nd round set of PCR-based indexing

2 index combination uniquely identi�es DNA strands from the same single-cell

sci-DNA-Seq

Single cell RNA barcoding and sequencing (SCRB-Seq)

SCRB-Seq AA(A)n

Single cell

Cell suspension

Cell sorting by FACS

Cell lysis Isolate RNA

AA(A)nAA(A)nT T (T)n

AA(A)nTT(T)n

Add adapters and reverse-transcribe

cDNAPool PCR

Cell labelUniversal primer

Oligo(dT)

Second strand RNA synthesis

Hybridize oligo

High-throughput single-cell labeling (Hi-SCL)

Hi-SCLBarcoded mRNA from single cells

AA(A)n

Singlecell

Cell suspension

Each droplet with unique oligos

Insert oligos in droplets

Load single cells into droplets with lysis bu�er

Fuse droplets Pool all droplets cDNA synthesis and ampli�cation

Sequence

Universal primer

Oligo(dT) RT bu�er

High-throughput single-cell labeling with indexing droplets (inDrop)

inDropBarcoded mRNA from single cells

AA(A)n

Singlecell

Cell suspension

Each microsphere with unique oligos

Oligos attached to hydrogel

Load single cells into droplets with lysis bu�er

Combine micro-spheres and droplets

Pool all droplets

UV primer release

Sequence

Photocleavable linker

Oligo(dT)RT bu�er

Cell label

A single nucleus RNA-Seq method (Nuc-Seq)

Nuc-Seq AA(A)n

Singlecell

Tissue Fixation and freeze

Lyse and centrifuge

Sort nuclei

Nuclei mRNA fragment

AAAAAA

cDNA synthesis TagmentationPCR

GGGLocked nucleic acid (LNA)

CCCGGG

Index 1Index 2

Gap repair and PCR

Single-cell RNA barcoding and sequencing (SCRB-Seq)

Div-Seq AA(A)n

Singlecell

Tissue in vivo labeled with 5-ethynyl-2’-de-oxyuridine (EdU)

Nuclei isolation

Click-IT tagging

FACS sort

mRNA fragment

AAAAAA

cDNA synthesis TagmentationPCR

GGGLocked nucleic acid (LNA)

CCCGGG

Index 1Index 2

Gap repair and PCR

CH3O CH3

Display methods on mobile device

Biotin

Preparation of acylated RNA for biotin–streptavidin puri�cation. DIBO, dibenzocyclooctyne

Biotin

Acylation

DIBO-biotin “click”

5-Ethynyl-2'-deoxyuridine (EdU)

5-iodouridine (5IU)

4-thiouridine (4SU)

5-bromo uridine (5BrU)

6-Thioguanosine (6SG)

Photoactivatable Nucleosides

Locked nucleic acid (LNA)

N NH 2

Cytosine 5-Methyl Cytosine

5-Methyl Cytosine

Uracil

Bisul�te conversion

N6-Methyladenosine (m6A)

Biotin

Biotin-4-�uorescein (B4F)

p-benzoylphenylalanine (Bpa)O

SUPeR-seq

Single-cell universal poly(A)-indepen-dent RNA sequencing (SUPeR-seq)

AA(A)nAAAAA

Add poly(A) primer with T7 promoter and PCR target

Reverse transcription and primer digestion with ExoSAP-IT

PCR ampli�cation Puri�cation DNA

AAAAANNNNNT T T T T

NNNNNT15NNNNNT15

NNNNNT15

AAAAAT T T T T

PolyA RNA

RT primer with well barcode and UMI

Transposase contains well-speci�c barcodes

AA(A)n

AAAARNA

AAAATTTT

Pool & sequence

Single-cell combinatorial indexing-based co-assay that jointly pro�les chromatin accessibility & mRNA (sci-CAR)

Nuclei are indexed for ATAC-Seq and RNA-Seq using well-speci�c barcodes

Addition of the 2nd well barcodes tags each nuclei with two di�erent combination of well barcodes, acting as a marker to contrast DNA fragments that are from di�erent cells

Open DNA

Single nuclei

Pool nuclei

Stain and FACS sort

2nd strand cDNA strand synthesis & nuclear lysis

Split wells into ATAC-Seq and RNA-Seq groups

Amplify DNA with RNA-Seq index

5000 nuclei/well

Add RNA-Seq index

Add ATAC-Seq index

Nuclei suspension

25 nuclei/well

in-situ tagmentation

DNA with ATAC-Seq index

DNA with RNA-Seq index

Seq adapters2nd well barcode

RNA-Seq dedicated lysate

Amplify DNA with ATAC-Seq index

DNA with ATAC-Seq index

DNA with RNA-Seq index

Seq adapters

2nd well barcode

ATAC-Seq dedicated lysate

sci-CAR

Integrated Techniques

Duplex-Seq α βVery rare mutation

Duplex sequencing detects rare mutations by sequencing and aligning both strands of the DNA

A mutation occurs on both strands

12 random base index

True variantRandom error

Ligate and PCR Rare variantSequence Create single strand consensus sequence from every unique molecular tag

ConsensusCreate duplex sequences based on molecular tags and sequencing primers

Add adapters

OS-Seq Gene

Target sequence

Adapter sequence

Flow cell

Sequencing Primers

Target sequence Single adapter library

Hybridize Hybridize

SequenceOligonucleotide-selective sequencing (OS-Seq) captures and sequence gene targets on the �ow cell

Create target-speci�c oligos Extend and Denature

Extend and Denature

Sequence reads 1 and 2

Fragment and add single adapters

Genome DNA and mRNA sequencing (DR-Seq)

DR-SeqAA(A)n

AA(A)n

polyA RNA

Single cell

RT with barcoded primerLyse cell Ad-2 primer

Split samples

Quasilinear ampli�cation

SequencegDNA ampli�cation

cDNA ampli�cationT T T T T T T T T TAAAAAAA

PCR and Remove adapters

2nd strand synthesis

Methylome and transcrip-tome sequencing from a single cell (scM&T-seq)

scM&T-Seq

Align RNA and methylome

AA(A)n

polyA RNA

Cell suspension

Isolate single cell

Separate the DNA and the RNALyse cell Sequence

T T T T T T T T T TAAAAAAA

Streptavidin magnetic bead with mRNA capture primerStreptavidin magnetic bead with mRNA capture primer

T T T T T T T T T TAAAAAAA On-bead transcriptome

ampli�cation with Smart-Seq2Whole-genome ampli�cation with scBS-seq

Very rare mutationSafe-SeqS

DNA Shear

Mutation

Amplify and solid phase capture

SequenceSafe-sequencing system is a unique molecular identi�er (UMI) approach to detect rare variants (Safe-SeqS)

Adapter ligation Randomly sheared ends serve as UMIs

Align sequences and determine actual ratio

True mutant

scChIP-Seq

Exonuclease digestion Immunoprecipitation DNADNA-protein complex DNA extraction

Crosslink proteins and DNA Sample fragmentationSingle cell chromatin immunoprecipitation (scChIP-seq)

Single-cell triple omics sequencing (scTrio-seq)

scTrio-SeqAA(A)n

DNA methylation

Cell suspension

Isolate single cell

Lyse and centrifuge

Supernatant

Nucleus

AA(A)npolyA RNA

Add carrier RNA

AA(A)nT T (T)ncDNA synthesis PCR and sequenceAdd poly A with TDTHybridize oligo

AA(A)n

DNAAdd sequencing adapters PCR and sequence

Align sequencesMethylated regions

Methylated adapter

End repair and ligation

Converted fragments

MspI digestion

PCR and sequence

Methylated DNA

scAba-SeqDNADetect 5hmC marks in single cells

with AbaSI nuclease (scAba-seq)Glucosylated 5-hmC

5hmc residues

T4-βGTHydroxy-methyl-ated DNA

AbaSI Ligate Pool T7 ampli�cationPrimer

Illumina 5’ adapterT7 promoter

Adapter with cell-speci�c barcodeSingle cell

Droplet-based single-cell ChIP-seq (Drop-ChIP)

Drop-ChIPSingle cell

Barcoded sequences from single cells

Cell suspension

Droplet with unique oligos

Load single cells into droplets with lysis bu�er and MNase

Fuse droplets Pool all droplets SequenceChromatin immuno-precipitation

Single cell

scATAC-Seq(Microfluidics)

Fragmented and primed DNASingle-cell assay for transposase accessible chromatin (scATAC-Seq)

Lyse and introduce Tn5 transposase

Pool libraries from all cells

Amplify with cell-speci�c barcodes

Insert in regions of open chromatinCell suspension

Micro�uidics device

Isolate single cell

scRC-Seq

Genomic DNA Enriched library

Novel retrotrans-position events

Retrotransposon binding site

Single cell retrotransposon capture sequencing (scRC-Seq)

Cell suspension

FACS isolation

Pick nuclei

Whole-genome ampli�cation

Create sequencing library

Sequence captureNucleus

Single cellscATAC-Seq(Cell index)

DNASingle-cell assay for transposase accessible chromatin (scATAC-Seq)

Barcode each well with Tn5 transposase

Cell suspension Isolate Nuclei Split sample

Pool and dilute

Split sample PCR-barcode every well

Pool for library prep

SMDBSingle-molecule droplet barcoding (SMDB)

DNA templates Single template encapsulation

Template ampli�cation Template fragmentation Barcode every droplet Pool for library prep

gRNA e�ciency

i5 i7UMIgRNA

UMI-based pooled CRIPSR screening for single-cell lineage tracing and quanti�cation of gRNA e�ciency (CRISPR-UMI)

Each gRNA construct contains random 10 nt UMI barcode, gene-speci�c spacer sequence, and Illumina indexed adaptors. They are also �anked by PacI restriction sites

Strong limiting dilution isolates di�erent editing outcomes to its own colonies

Each cell in a colony was edited by speci�c gRNA hence contains the same UMI sequence

gRNA sequence is read and resulting data used to determine which gRNAs were most e�cient in creating the desired outcome

CRISPR/Cas9Cell suspension

CRISPR-UMI gRNA design

Edit, select, expand Expand Purify DNA

and amplify

gRNA constructsLimiting

dilution

CRISPR-UMI

Single-cell dissection and UV catapulting into collection tubes

Topographic single-cell sequencing (TSCS)

Tissue imaging to capture spatial informa-tion

Single-cell whole-genome ampli�cation by DOP-PCR

Barcoded sequencing libraries

Each single-cell tagged by unique barcodes

Spatial information can be traced back using the barcodes

Single-cells in collection tubes

DOP-PCRCell-speci�c barcodes

DNA with spatial info

Frozen tissue slices

UV-catapulting Cell-speci�c barcodes

TSCSSingle cell

Random primer

Methylated DNA

Amplify and sequence

Single-nucleus methyl-cytosine sequencing (snmC-Seq)

Isolated single cell

Random priming Extend Pool samples and Adaptase reaction

AdaptersnmC-Seq

Methylated DNASingle cell

Isolate nuclei and nucleosome depletion

Pool Bisul�te conversion, linear ampli�cation, and PCR ampli�cation

Transposase-based tagging

Random sorting

Transposome

PCR-based tagging

Random sorting

Single-cell combinatorial indexed for methylation analysis (sci-MET)

Tagmented with cytosine-deplet-ed adapters. Adapters contain well-speci�c index and read 1 primer sequence

This step converts unmethylated cytosines, tags fragments with a second well-speci�c barcode, and attaches sequencing adapters.

1st adapterSequencing adapters + well-speci�c index

sci-MET

Cell barcodeT7 promoter

Tn5 bind sitePool

Pool &sequence

Single nuclei

Single-cell transposome hyper-sensitive site sequencing (scTHS-Seq)

SplitSplit

In-vitro transcription ampli�cation

3’ end transposition & end �ll-in

Sequencing adaptor ligation & amplify

Nuclei suspensionOpen DNA

2000 nuclei/well

100 nuclei/well

3’ adaptors Illumina indexed adaptors

Tn5059 transposome

scTHS-seq

Single cell

Release nuclei and Dpn II digest

Ligation of 1st barcode

Ligation of 2nd barcode

Ligation of barcoded bitotinylated double-stranded bridge adaptors

Proximity ligation

Ligation of custom-barcoded Y-adaptors with sequencing adaptors

PCR amplify and sequence

SplitFixed cells Well-plate

Protein-DNA complex

1st barcode

2nd barcode

Pool Pool & purifySplit

Single-cell combinatorial indexed Hi-C (sciHi-C)

Biotin

At most 25 cells per well

sciHi-C

Genome and transcriptome sequencing from a single cell (G&T-seq)

G&T-Seq

Align RNA and genome

AA(A)n

polyA RNA

Cell suspension

Isolate single cell

Separate the DNA and the RNALyse cell Sequence

T T T T T T T T T TAAAAAAA

Streptavidin magnetic bead with mRNA capture primer

ampli�cation with Smart-Seq2Whole-genome ampli�cation with MDA

AA(A)n

polyA RNA

Copy Number Alterations

DNA methylation

scBS-Seq

Supernatant

Nucleus

AA(A)npolyA RNA

Add carrier RNA

AA(A)nT T (T)ncDNA synthesis PCR and sequenceAdd poly A with TDTHybridize oligo

AA(A)n

DNAAdd sequencing adaptors PCR and sequence

Cellsuspension

Lyse and separate RNA from nucleus using magnetic bead and centrifugation

Single-cell triple omics sequencing version 2 (scTrio-Seq2)

Random primer 1

Methylated DNA Bisul�te conversion

Random primer 2

Align fragments from every UMI and sequence

First random priming

Second random priming

Repeat 4 times

PCRExtend

Adapter AdapterExo I and purify

Single cellscTrio-Seq2

CpG dinucleotides Methylated CpG

Single-cell nucleosome, methylation and transcription sequencing (scNMT-Seq)

Isolate single cell

Lyse and GpC methylase labelling

AA(A)nSingle cell

polyA RNA

DNA methylation

Chromatin accessibility

Transcriptome

DNA methylation

Chromatin accessibility

DNA T T T T T T T T T TAAAAAAA

Streptavidin magnetic bead with mRNA capture primer

Methylated CpG indicates accessible DNA

Isolate mRNA

ampli�cation with Smart-seq2

Whole genome bisul�te sequencing with scBS-seq

scNMT-seq

Biotin

6 rounds of random priming with biotinylated adapters

Methylated GpC dinucleotides mark the absence of nucleosomes

Capture �rst strand on Streptavi-din-coated magnetic beads

Streptavidin

2nd random primer

Amplify and sequence

Methylated CpGdinucleotides

Methylated GpCdinucleotides

GpC methyl-transferaseLyse

Chromatin

NOMe-Seq PBAT

Chromatin overall omic-scale landscape sequencing (scCOOL-Seq)

Single cell

DNA methylation

CNV and ploidy

Chromatin accessibilityand nucleosome position

scCool-Seq

Sort cells to microplate wells Hypotonic lysis

Physical separation

SupernatantLibrary prep

Nuclei

Total RNA

From cellular mRNA

From genomic DNA

AntibodySurface antigen

Microwell

Magnetic microbeadDNA DNA

Simultaneous isolation of genomic DNA & total RNA from single-cells (SIDR)

Bead-binding prior to cell lysis reduces the number of cell-surface proteins that are solubilized

Hypotonic lysis releases cytoplasmic material but preserves the integrity of the nuclear membrane

Nuclei remain encapsulated by semi-per-meabilized cell membranes and can be isolated magnetically”

cDNA libraries are ready for sequencing

AA(A)nNon-polyA RNA

PolyA RNA

SIDRSingle cell

Crosslinked chromatin

Tagmented DNA

Restriction digest

Single cell

Fixed cell

Single-cell chromatin conformation capture method with multiplex end-tagging ampli�cation (Dip-C)

DNA close to each other in proximity are digested from chromatin and ligated to each other using DNA ligase. The removal of all biotin-pull-downsteps increases e�ciency

20 di�erent sequences of META barcodes reduce the amount of DNA lost due to fragments having the same sequencing tags to 1/20 of input DNA. PCR-based seq adapter addition also reduce arti�cial chimeras

Sophisticated algorithm can also identify chromosomal haplotypes linked by contact

META transposome

TransposaseSeq adapters

META PCR primers

META barcodesDNA

META PCR primer20 bp META barcode

DNA ligase

Lyse cell

Amplify with META primers

For Research Use Only. Not for use in diagnostic procedures.

© 2020 Illumina, Inc. All rights reserved. Illumina, Inc. • 5200 Illumina Way, San Diego, CA 92122 USA • 1.800.809.4566 toll-free • 1.858.202.4566 tel • techsupport@illumina.com • illumina.comIllumina, HiSeq, MiSeq, MiniSeq, Nextera, NextSeq, TruSeq, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No. 770-2020-002-A QB9466. Current as of 14 April 2020.

This poster was compiled by the Illumina Scienti�c A�airs. Additional information, the latest version of the poster, and a comprehensive list of *seq methods, are available at http://www.illumina.com/libraryprepmethods. Please contact Scienti�c A�airs with any questions, comments, or suggestions.

ReferencesAct-Seq Wu Y. E. et al. (2017) Neuron 96(2): 313-329CEL-Seq Hashimshony T. et al. (2012) Cell Rep 2: 666-673CirSeq Acevedo A. et al. (2014) Nature 505: 686-690CITE-Seq Stoeckius M., et al. (2017) Nat Methods 14(9): 865-868CLaP Binan L. et al. (2016) Nat Commun 7: 11636CRISPR-UMI Michlits G. et al. (2017) Nat Methods 14(12): 1191-1197CROP-Seq Datlinger P. et al. (2017) Nat Methods 14(3): 297-301CytoSeq Fan H. C. et al. (2015) Science 347: 1258367Digital RNA Shiroguchi K. et al. (2012) Proc Natl Acad Sci USA

109:1347-1352Dip-C Tan L., et al. (2018) Science 361(6405): 924-928Div-Seq Habib N. et al. (2016) Science 353(6302): 925-928DP-Seq Bhargava V. et al. (2013) Sci Rep 3: 1740

DroNC-seq Habib N. et al. (2017) Nat Methods 14(10): 955-958Drop-Seq Macosko E. Z. et al. (2015) Cell 161: 1202-1214DR-Seq Dey S. S. et al. (2015) Nat Biotechnol 33: 285-9Drop-ChIP Rotem A. et al. (2015) Nat Biotechnol 33: 1165-72Duplex-Seq Schmitt M. W. et al. (2012) Proc Natl Acad Sci USA 109:

14508-14513ECCITE-seq Mimitou E. P. et al. (2019) Nat Methods 16(5): 409-412FREQ-Seq Chubiz L. M. et al. (2012) PLoS One 7: e47959FRISCR Thomsen E. R. et al. (2016) Nat Methods 13: 87-93G&T-seq Macaulay I. C. et al. (2015) Nat Methods 12: 519-522HiRes-Seq Imashimizu M. et al. (2013) Nucleic Acids Res 41:

9090-9104Hi-SCL Rotem A. et al. (2015) PLoS One 10: e0116328

IMS-MDA Seth-Smith H. M. et al. (2013) Nat Protoc 8: 2404-2412inDrop Klein A. M. et al. (2015) Cell 161: 1187-201LIANTI Chen C. et al. (2017) Science 356(6334): 189-194MALBAC Zong C. et al. (2012) Science 338: 1622-1626MARS-seq Jaitin D. A. et al. (2014) Science 343:776-9MATQ-seq Sheng K. et al. (2017) Nat Methods 14(3): 267-270MDA Dean F. B. et al. (2001) Genome Res 11: 1095-1099Microwell-seq Han X. et al. (2018) Cell 172(5): 1091-1107.e1017MIDAS Gole J. et al. (2013) Nat Biotechnol 31:1126-32MIPSTR Carlson K. D. et al. (2015) Genome Res 25: 750-761Mosaic-seq Han X. et al. (2018) Cell 172(5): 1091-1107 e1017MULTI-seq McGinnis C. S. et al. (2019) Nat Methods 16(7): 619-626NanoCAGE Plessy C. et al. (2010) Nat Methods 7: 528-534

NanogridSNRS Gao R. et al. (2017) Nat Commun 8(1): 228nuc-seq Wang Y. et al. (2014) Nature 512: 155-160Nuc-Seq/SNES Leung M. L. et al. (2015) Genome Biology 16(1): 55OS-Seq Myllykangas S. et al. (2011) Nat Biotechnol 29: 1024-1027PAIR Bell T. J. et al. (2015) Methods Mol Biol 1324: 457-68Quartz-Seq Sasagawa Y. et al. (2013) Genome Biol 14: R31Quartz-Seq2 Sasagawa Y. et al. (2018) Genome Biology 19(1): 29RamDA-seq Hayashi T. et al. (2018) Nature Communications 9(1): 619RNAtag-Seq Shishkin A. A. et al. (2015) Nat Methods 12: 323-325Safe-SeqS Kinde I. et al. (2011) Proc Natl Acad Sci USA 108: 9530-5scABA-seq Mooijman D. et al. (2016) Nature Biotechnology 34: 852scATAC-seq Buenrostro J. D. et al. (2015) Nature 523: 486-490 (Microfluidics)

scATAC-Seq Cusanovich D. A. et al. (2015) Science 348: 910-4 (Cell Index)scChip-seq Rotem A. et al. (2015) Nat Biotechnol 33: 1165-72scCool-seq Li L. et al. (2018) Nature Cell Biology 20(7): 847-858sciHi-C Ramani V. et al. (2017) Nature Methods 14: 263sci-CAR Cao J. et al. (2018) Science 361(6409): 1380sci-DNA-seq Rosenberg A. B. et al. (2018) Science 360: 176-182sci-MET Mulqueen R. M. et al. (2018) Nature Biotechnology 36: 428sci-RNA-seq Cao J. et al. (2017) Science 357(6352): 661SCMDA Dong X. et al. (2017) Nature Methods 14: 491scM&T-seq Angermueller C. et al. (2016) Nature Methods 13: 229scNMT-seq Clark S. J. et al. (2018) Nature Communications 9(1): 781scRC-Seq Upton K. R. et al. (2015) Cell 161: 228-39scRNA-seq Tang F. et al. (2009) Nat Methods 6: 377-82

SCRB-Seq Soumillon M. et al. (2014) bioRxiv: 003236scTHS-seq Lake B. B. et al. (2018) Nature Biotechnology 36(1): 70-80scTrio-seq Hou Y. et al. (2016) Cell Res 26: 304-19scTrio-seq2 Bian S. et al. (2018) Science 362(6418): 1060Seq-Well Gierahn T. M., et al. (2017). Nat Methods 14(4): 395-398SIDR Han K. Y. et al. (2018) Genome Research 28(1): 75-87SINC-seq Abdelmoez M. N. et al. (2018) Genome Biology 19(1): 66Smart-Seq Ramskold D. et al. (2012) Nat Biotechnol 30: 777-782Smart-seq2 Picelli S. et al. (2013) Nat Methods 10: 1096-1098vSMDB Lan F. et al. (2016) Nat Commun 7: 11784smMIP Hiatt J. B. et al. (2013) Genome Res 23: 843-854snDrop-seq Lake B. B. et al. (2018) Nature Biotechnology 36(1): 70-80SNES Leung M. L. et al. (2015) Genome Biol 16: 55

snmC-Seq Luo C. et al. (2017) Science 357(6351): 600snRNA-seq Grindberg R. V. et al. (2013) Proc Natl Acad Sci USA 110:

19802-7SPLiT-seq Rosenberg A. B. et al. (2018) Science 360(6385): 176STRT Islam S. et al. (2011) Genome Res 21: 1160-1167SUPeR-seq Fan X. et al. (2015) Genome Biol 16: 148TCR Chain Pairing Turchaninova M. A. et al. (2013) Eur J Immunol 43:

2507-2515TCR-LA-MC-PCR Ruggiero E. et al. (2015) Nat Commun 6: 8081TIVA Lovatt D. et al. (2014) Nat Methods 11: 190-196TSCS Casasent A. K. et al. (2018) Cell 172(1): 205-217.e212UMI Method Kivioja T. et al. (2012) Nat Methods 9: 72-74viscRNA-seq Zanini F. et al. (2018) Elife 7: e32942

Sequencing by Synthesis

TruSeq™ PCR Free

Double-stranded DNA

FractionateSize select

A-overhang

End repairPhosphorylate

IndexT

Index Adapter ligation

P5P7Index

Add Adapters

Product ready for cluster generation

TruSeq™ NanoDouble-stranded DNA

FractionateSize select

A-overhang

End repairPhosphorylate

Index 1Index 2

Index 2Index 1

Adapter ligation

Denature and amplify

Add Adapters

Index 1Index 2

Index 2Index 1

P5 P7Index 1Index 2

Double-stranded DNA

TruSeq™ Small RNA

3’5’ Small RNA fragment

Ligate adapters

Add primer

5’ Adapter 3’ Adapter

Index 1

P5 P7Index

AATTCGC

Synthesize second strand

The second read is sequenced

Sequence Index2

AATTCGC

Deblock P5 primer and add unlabeled bases

Read 2 primer

The forward-strand is cleaved and washed away

AATTCGC

Adapter hybrid-izes to flowcell

Reverse strand synthesis

Reverse strand

Forward strand

Remove forward strand

Fold over and hybridize to second primer

Synthesize second strand

The reverse strand is cleaved and washed away

With each cycle, four fluores-cently tagged nucleotides compete for addition to the growing chain. Only one is incorporated based on the sequence of the template.

The read product is washed away

Thousands of molecules are amplified in parallel

Reverse strand

Forward strand

Bridge amplification

Sequence primer

Fold over and hybridize to first primer

Sequence Index1

Index 1 primer

The read product is washed away

TruSeq™ RNA Exome

Target

TargetP5 P7

Index 1Index 2 Product ready for cluster generation

Pool stranded RNA-Seq libraries

Biotinylated target probe

Hybridize probes to targets

Capture on streptavidin magnetic beads

TruSeq™ Targeted RNA ExpressionTarget

ULSO DLSO

Total RNA

Hybridization

Index 1

5’ P

Index 2

Target Index 1Index 2 Product ready for cluster generation

Add custom primers

Extension-Ligation

Nextera™ Library Preparation

Transposase

~300bp

Tagmentation

Ampli�cation

P5 P7Index 1Index 2

Index 2

Index 1

Nextera™ Mate Pair

Adapter ligation

Isolate biotinylatedfragment

Transposase

Tagmentation

Circularize

R R R RBiotinylated junction adapter

Fragment

Nextera™ Rapid Capture

Target

TargetP5 P7

Index 1Index 2 Product ready for cluster generation

Denatured and pooled fragments from Nextera library

Capture onstreptavidin magnetic beads

Hybridize probes to targets

Biotinylated target probe

AmpliSeq™ for Illumina

Remove primer sequences

Add sequencing primers

Index 2Index 1

Index 1P7

Index 2P5 Product ready for

cluster generation

DNA/cDNA

DNA RNA5’ 3’

Reversetranscribe

Ligate adaptors

TruSeq™ RNA

Total RNA

T T T T TAAAAA polyA select

Fragment

Random hexamer

First and second strand synthesis

5’ 3’

TruSeq™ Stranded RNARNA/mRNARandom primer

Create cDNA

Create second strand cDNA

End repairPhosphorylateA-overhang

Adaptor ligation

dUTP + dCTP + dATP + dGTP

dT TP + dCTP + dATP + dGTP

P5 P7Index 1Index 2

U U U UUUUUUUUSense strand

PU U U UUUUUUUSense strand

Index 1

P5Index 2

U U U UUUUUUU Index 2

Index 1

Sense strand

P5P7U U U UUUUUUU

Sense strand Block polymerase

5’ 3’ AAAAA

SureCell™ WTA 3’

Ampli�cation

3’ enrichment and sample indexing

Direct cDNA Nextera tagmentation

Cell lysismRNA hybridization

Single cells encapsu-lated in droplets

cDNA synthesis and barcoding

Barcoded beads

UMI Barcode Read1T T T T TAAAAA

mRNAUMI Barcode Read1

T T T T T

mRNAAAAAA

UMI Barcode Read1T T T T T

mRNAAAAAA

IndexP7 P5

UMI Barcode Read1

Technology Poster Single-Cell PB 2020 DPI 041420 VIEW · AAAAA mRNA UMI Barcode Read1 TTT mRNA...

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