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Key Yellow highlights indicate the target of the protocol Single-Cell For all you seq... RNA Low-Level Detection DP-Seq Designed Primer-based RNA-se- quencing strategy (DP-seq) DNA cDNA AA(A) n Define set of heptamer primers Poly(A) selection First strand cDNA synthesis Hybridize primers PCR AA(A) n TT(T) n No secondary structure Unique sequence AA(A) n TT(T) n Digital RNA HiRes-Seq FREQ-Seq RNAtag-Seq cDNA cDNA1 cDNA2 cDNA1 cDNA2 Amplify Sequence Unique 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 cDNA1 cDNA2 mRNA Smart-Seq NanoCAGE AAAAAAA mRNA fragment AAAAAAA Second strand synthesis AAAAAA TTTTTT DNA TTTTTT Adapter Adapter Switch mechanism at the 5’ end of RNA templates (Smart) PCR amplification Purify First-strand synthesis with MMLV reverse transcriptase CCC CCC mRNA UMI Method AAAAAAA mRNA fragment AAAAAAA First strand synthesis Second strand synthesis AAAAAA TTTTTT P7 True variant Random error DNA TTTTTT P5 Index Degenerate molecular tag (N10) Unique molecular identifiers (UMIs) uniquely identify copies derived from each molecule PCR amplification Align fragments from every unique molecular tag CCC CCC mRNA Smart-Seq2 AAAAAAA mRNA fragment AAAAAA cDNA synthesis Tagmentation AAAAAA AAAAAA TTTTTT TTTTTT Adapter Switch mechanism at the 5’ end of RNA templates (Smart) PCR First-strand synthesis with MMLV reverse transcriptase CCC CCC GGG Tem- plate-switch- ing oligo Locked nucleic acid (LNA) CCC GGG Enrichment-ready fragment P5 P7 Index 1 Index 2 Gap repair, enrich- ment PCR and PCR purification STRT Single-cell tagged reverse transcription (STRT) AA(A) n AA(A) n AA(A) n AA(A) n AA(A) n AA(A) n Cell 1 Cell 2 Cell 3 TT(T) n TT(T) n TT(T) n AA(A) n AA(A) n AA(A) n TT(T) n TT(T) n TT(T) n CCC CCC CCC cDNA synthesis Add 3 to 6 cytosines TT(T) n TT(T) n CCC CCC CCC GGG GGG GGG 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 1 TT(T) n Unique index 5’ adapter GGG CEL-Seq AA(A) n AA(A) n AA(A) n AA(A) n AA(A) n AA(A) n AA(A) n AA(A) n AA(A) n Cell 1 Cell 2 Cell 3 T7promoter Unique index 5’ adapter TT(T) n TT(T) n TT(T) n TT(T) n AA(A) n AA(A) n AA(A) n TT(T) n TT(T) n TT(T) n Second strand RNA synthesis Fragment, add adapters and reverse-transcribe Separate cell sequences based on unique indices Pool Cell 3 Cell 2 Cell 1 Cell expression by linear amplifica- tion and sequencing (CEL-Seq) PCR cDNA synthesis Tagmentation PCR First strand synthesis AAAAAA TTTTTT Adapter CCC AAAAAA TTTTTT CCC GGG CCC GGG Enrichment-ready fragment P5 P7 Index 1 Index 2 Gap repair and PCR Single-nuclei RNA sequencing (snRNA-seq) snRNA-Seq AA(A) n Single cell polyA RNA Cell suspension Lyse and centrifuge Sort nuclei Supernatant Nuclei Nucleus cDNA synthesis Tagmentation PCR First-strand synthesis AAAAAA TTTTTT Adapter CCC AAAAAA TTTTTT CCC GGG CCC GGG Enrichment-ready fragment P5 P7 Index 1 Index 2 Gap repair and PCR Fixed and recovered intact single-cell RNA (FRISCR) FRISCR AA(A) n Fixed single cell polyA RNA Cell suspension Fix Sort single cells Isolate RNA Lyse cells and reverse crosslink AAAAAA Quartz-Seq Whole-transcript amplifi- cation for single cells (Quartz-Seq) AA(A) n AAAAA AAAAA TTTTT TTTTT T7 PCR Add poly(A) primer with T7 promoter and PCR target AAAAA TTTTT Reverse transcription and 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 TTTTT PCR TTTTT T7 PCR AAAAA TTTTT PCR AAAAA TTTTT T7 PCR AAAAA Blocking primer with LNA cDNA MARS-Seq Massively parallel RNA single-cell sequencing framework (MARS-Seq) AA(A) n AAAAA TTTTT T7 UMI Add poly(A) primer with partial T7 promoter and UMI Second strand synthesis RNA fragmentation RNA to ssDNA ligation DNaseI Reverse transcription PCR and purification cDNA AAAAA TTTTT T7 UMI partial rd1 rev P5 P7 scRNA-seq Single-cell mRNA sequencing (scRNA-seq) AA(A) n AAAAA Add polyT primer Reverse-transcribe Poly(A)-taile d mRNA Reverse transcription and primer digestion with ExoSAP-IT PCR amplification Shear DNA AAAAA TTTTT TTTTT AAAAA TTTTT TTTTT AAAAA TTTTT TTTTT AAAAA Corrected sequence Identify low abundance RNA viruses with circular sequencing (CirSeq) CirSeq Whole-genome RNA AA(A) n Circularize RNA with kinase and RNA ligase 1 Random primers Circular RNA template Repeat 1 Repeat 2 Repeat 3 Repeat 1 Repeat 2 Repeat 3 Mutation Error Transcriptome in vivo analysis (TIVA) TIVA Whole-genome RNA AA(A) n Capture on Streptavidin coated magnetic beads mRNA from single cell AAAAAAA AAAAAAA UUUUUUUUUUUUUUUUUU Cy3 Cy5 PL CPP Biotin PL S S CPP Cell-penetrating peptide Disulfide bond S S Photocleavable linker PL UUUUUUUUUUUUUUUUUU Cy3 UUUUUUUUUUUUUUUUUU Cy3 CPP S AAAAAAA AAAAAAA UUUUUUUUUUUUUUUUUU Cy3 Cy5 PL PL S UUUUUUUUUUUUUUUUUU Cy3 AAAAAAA AAAAAAA Cy5 PL PL S AAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAA Load into cells CPP peptide released Photoactivate Anneal to mRNA Cell Gene expression cytometry (CytoSeq) CytoSeq Barcoded mRNA from single cells AA(A) n Single cell Cell suspension Each bead with unique oligos Load cells and beads into microwells Cell lysis, mRNAs hybridize on bead Pool all beads from microwells cDNA synthesis and amplification Sequence Universal Cell label Molecular index Oligo(dT) Analyze mRNA transcripts from individual cells in droplets (Drop-seq) Drop-Seq Barcoded mRNA from single cells AA(A) n Single cell Cell suspension Each bead with unique oligos Load cells and beads into droplets Cell lysis, mRNAs hybridize on bead Pool all beads from droplets cDNA synthesis and amplification Sequence Universal Cell label Molecular index Oligo(dT) TCRα mRNA TCRβ mRNA Oil emulsion Identify T-cell Receptor (TCR) alpha–beta chain pairing in single cells Reverse transcription Amplification Overlap extension Blocker primers Nested PCR amplification TCR Chain Paring AA(A) n AA(A) n TCRα TCRβ TCRα TCRβ TCRα TCRβ TCRα TCRβ TCRα TCRβ DNA PCR suppression of non-fused molecules CDR3α CDR3β CDR3 Whole-genome RNA Peptide nucleic acid-assisted identification of RNA binding protein (PAIR) PAIR Capture on magnetic beads Visualize protein on SDS-PAGE CPP Cell-penetrating peptide Disulfide bond S S Photo-activatible compound Bpa Binding site for RNA-bind- ing protein Create peptide nucleic acid analogs (PNAs) AA(A) n AA(A) n PNA CPP S S PNA Bpa CPP peptide released Bpa CPP S PNA Bpa S AA(A) n AA(A) n S S Bpa Photoactivate Load into cells AA(A) n Cell labeling via photobleach- ing (CLaP) CLaP Barcoded mRNA from single cells AA(A) n Single cell Confluent cells in culture Biotin-4-fluo- rescein (B4F) Photobleach and crosslink with 473 nm laser Cy5-streptavidin labeling Tagged cells isolated, reverse-transcribed and sequenced Rinse 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 PCR 1 C RNA C P5 P7 Index 1 Index 2 PCR 2 with nested primers Add sequencing adapters DNA ready for sequencing CCCCC CCCCC CCCCC CCCCC MALBAC primers RNA UMI cDNA PolyA RNA Single cell Non-polyA RNA Reverse transcription RNA digestion and dC tailing G-enriched MALBAC primers with UMIs GGGGG GGGGG CCCCC Cell lysis Repeat 9x Multiple annealing and dC-tailing-based quantita- tive scRNA-Seq (MATQ-Seq) MALBAC primers initiate quasilinear amplification to reduce amplification of PCR bias Primer hybridization at low temperatures increases RT efficiency and non-polyA transcript detection Addition of dC tail to 1st cDNA strands increases efficiency of 2nd strand synthesis UMIs drastically reduce exponential PCR amplification bias Library prep and sequence AA(A) n MATQ-seq Random displacement amplification sequencing (RamDA-Seq) Strand displacement amplification 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 cDNA 3’ 5’ DNase I gp32 Non-polyA RNA PolyA RNA NSR primer Cell lysis Single cell NSR primer RamDA-seq AA(A) n AA(A) n Single cell Single cell Nuclei Microchannel containing nuclei Microchannel containing cytoplasm Cytoplasm Lysis and ITP extraction Load into microfluidics system ITP acceleration isolates cytRNA nucRNA-Seq by SMART-Seq2 Custom microfluidics with isotachophoresis (ITP) buffer chemistry cytRNA-Seq by SMART-Seq2 Nuclear RNA Cytoplasmic RNA Sequencing-ready cDNA 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-specific 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 RNA AA(A) n Infected cells FACS sort into well plates Lyse RT and template-switching Amplify and library prep Capture RNA Modified SMART-Seq2 viscRNA-seq AAAAA TTTTT AAAAA TTTTT AAAAA TTTTT TTTTT PolyA RNA AA(A) n Single-cell in PCR well plate Cell barcode UMI cDNA mRNA PCR 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 effectively uses limited sequence reads (Quartz-Seq2) Single cells are sorted into PCR well plates containing lysis buffer 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 efficiency Custom library prep attaches sequencing adapters and pool barcodes to enable mixing of different sets of cell barcode-labeled cDNA Quartz-seq2 Fixed cells RNA cDNA Barcode 1 Barcode 2 Barcode 3 Barcode 4 Pool RT with 1st barcode set Ligate 2nd barcode set Ligate 3rd barcode set Amplify with 4th barcode set PCR of cDNA using 4th well-specific barcode along with sequenc- ing adaptors Fixed and permeabilized cells are reverse-tran- scribed using microwell- specific 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 split Random split Pool Pool Biotin Single cell Split-pool ligation-based transcriptome sequencing (SPLiT-Seq) PolyA RNA AA(A) n SPLiT-seq UMI 2nd index tagging and sequencing adapters Well-specific transposomes i5 adapter with cell index i7 adapter with cell index Pool Index 1 cDNA Index 2 UMI Single 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-specific index cDNA strands are tagged with another set of well-specific barcodes that includes i7 and i5 sequencing adapters Ready for sequencing RT with 1st barcode set Fixed cells Random split Random split Pool 2nd strand synthesis PolyA RNA AA(A) n TTTT TTTT TTTT sci-RNA- Seq Each bead has unique cell-identifying oligo sequence Load nuclei and beads into droplets Break droplets & pool beads cDNA synthesis and amplification Oligobeads Droplets Droplets (enlarged) Sequencing-ready cDNA PCR handle Cell barcode UMI Oligo(dT) AAAA AAAA AAAA mRNA Single nuclei Load nuclei into droplet microfluidic system Single nuclei droplet-based sequencing (snDrop-Seq) PolyA RNA Nuclei suspension Single nuclei AA(A) n Nuclei lysis snDrop-Seq Massively parallel single-nucleus RNA sequencing with droplet technology (DroNc-seq) AA(A) n Cell suspension Each bead has unique cell-identifying oligo sequence Load cells and beads into droplets Barcoded mRNA from single nuclei Pool all beads from droplets cDNA synthesis and amplification Sequence Lyse PolyA RNA Oligobeads PCR handle Cell barcode UMI Oligo(dT) Single nuclei Isolate nuclei AAAA AAAA AAAA mRNA non-polyA RNA DroNC-Seq DNA Low-Level Detection MALBAC Genome Hybridize primers PCR 27-bp common sequence 8 random nucleotides Partial amplicons Template Denature Denature Hybridize primers Synthesis Multiple annealing and looping-based amplification cycles (MALBAC) DNA Cycles of quasilinear amplification Looped full amplicons Bst DNA polymerase Genomic DNA Gene smMIP Copy target sequence Exonuclease Corrected sequence Align fragments from every unique molecular tag Sample index Read1 Read2 True variant Random error Single Molecule Molecular Inversion Probes (smMIPs) for detecting low frequency targets PCR amplification Degenerate molecular tag Targeted STR Short tandem repeat (STR) MIPSTR Copy target STR Amplify and sequence Targeted capture of STR loci by smMIPs (MIPSTR) Degenerate molecular tag Strain I Strain II Strain I Strain I Natural variation between individuals Somatic variation within an individual Nuc-Seq SNES Cell 1 Cell 2 Cell 3 Cell sorting from G2/M distribution Lyse cell Nucleus Single G2/M nucleus sequencing of cells in S phase (nuc-seq) Single nucleus exome sequencing (SNES) Single cell genome phi29 Limited amplification S1 nuclease Synthesis DNA Genome MDA IMS-MDA MIDAS Primer hybridization Nascent replication fork phi29 phi29 S1 nuclease Amplified DNA 3’ blocked random hexamer primers Synthesis Synthesis Multiple displacement amplification (MDA) Immunomagnetic separation for targeted bacterial enrichment for MDA (IMS-MDA) Microwell displacement amplification 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-specific transcripts are identified by barcode sequences and couple gene expression data with cell-surface protein marker data Cellular indexing of transcriptomes & epitopes by sequenc- ing (CITE-Seq) Antibody Antibody barcode Single cell Cells Load into droplets PolyA RNA Surface protein AA(A) n AAAA AAAA AAAA AA(A) n SS Oligobeads Antibody-oligo complex PCR handle Cell barcode UMI Oligo(dT) mRNA 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 identified 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 Antibody Antibody barcode AA(A) n SS Antibody-derived tags Antibody AA(A) n Sample A barcode Antibody AA(A) n Sample B barcode Antibody AA(A) n Sample C barcode Cell lysis AAAA AAAA AAAA mRNA 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-specific tagging Droplet microfuildics Pool Expanded CRISPR-compatible cellular indexing of transcriptomes and epitopes by sequencing (ECCITE-Seq) Hashtag oligos (HTOs) target specific proteins with barcodes to provide sequence-based cellular identification of sample origin. Antibody-derived tags mark cellular antigens of interest gRNA ECCITE-Seq PCR handle Cell barcode UMI Oligo(dT) AA(A) n AAAA AAAA AAAA PolyA RNA gRNA CRISPR/Cas9 Single cell Cell suspension gRNA construct Drop-Seq Lentiviral transduction & enrichment AAAA AAAA AAAA AAAA mRNA gRNA gRNA Transcriptome Each bead has unique cell-identifying oligo sequence Puromycin selection enriches successful gRNA-transduced cells Load cells and beads into droplets scRNA-seq directly link transcriptome profile with corresponding guide RNA Lyse cDNA library Break 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 PCR handle Cell barcode UMI Oligo(dT) AA(A) n AAAA AAAA AAAA PolyA RNA gRNA CRISPR/Cas9 Single cell Cell suspension gRNA construct Viral infection & enrichment gRNA Transcriptome Puromycin selection enriches successful gRNA induced cells scRNA-seq directly link transcriptome profile with corresponding guide RNA cDNA library Break 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 AAAA AAAA AAAA AAAA mRNA gRNA Each bead has unique cell-identifying oligo sequence Load cells and beads into droplets Lyse Oligobeads PCR handle Cell barcode UMI Oligo(dT) Minimize artificially induced transcrip- tional perturbations followed by scRNA-Seq (Act-Seq) Actinomycin D (ActD) inhibits dissocia- tion-induced gene expression common in conventional dissociation methods Each bead has unique cell-identifying oligo sequence Load nuclei and beads into droplets Break droplets & pool beads cDNA synthesis and amplification Oligobeads Droplets Droplets (enlarged) Sequencing-ready cDNA PCR handle Cell barcode UMI Oligo(dT) AAAA AAAA AAAA mRNA Load into droplet microfluidic system Single cell Cell lysis polyA RNA Single cell Frozen tissue slice Drop-Seq Cell dissociation followed by Actino- mycin D treatment AA(A) n Act-Seq PDMS subnanoliter array Oligobeads Cell suspension mRNA 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 Single cell AA(A) n Universal Cell label Molecular index Oligo(dT) Seq-Well Agarose microwell array Oligobeads Cell suspension Single-cell mRNA mRNA Adaptors Transcriptomic profiles 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 Single cell AA(A) n UMI Cell label Molecular index Oligo(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 fluorescence imaging Fluorescent microscopy imaging polyA RNA Single cell or nuclei AA(A) n Nanogrid Oligo-lined nanowells None/dead in well More than 1 per well 1 nucleus/cell per well DAPI-stained nuclei Dispense Select viable wells & lyse RT, template switching, and one-sided P7 tagmentation Barcoded cDNA library flanked by sequencing adapters cDNA library generation P5 adapter Well barcode UMI Oligo(dT) TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT TTTT mRNA cDNA Nanogrid SNRS Pool scRNA-Seq workflow Single cell Cell cultures with different treatments LMO-labeled cells Lipid-modified oligonucleotide (LMO) with barcode LMO sample barcodes are identical within a treatment group but unique among different 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 mRNA AA(A) n AAAA AAAA MULTI-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 amplification (SCMDA) with single-cell variant caller (SCcaller) SCMDA Linear amplification 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 amplification of gDNA significantly reduces amplifica- tion bias and replication errors RT RNase digestion and 2nd strand cDNA synthesis and UMI barcoding Genome gDNA gDNA RNA cDNA UMI cDNA/RNA hybrid LIANTI Transposome Lyse Transposase T7 RNA Polymerase Transposase bind site T7 Promoter Single cell LIANTI Pool Pool Transposase-based tagging Random sorting Isolate nuclei and nucleosome depletion Transposome DNA DNA Single 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-specific unique indexed adapters 15-25 cells are randomly sorted into wells for 2nd round set of PCR-based indexing 2 index combination uniquely identifies 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) n AA(A) n T T (T) n AA(A) n TT(T) n Add adapters and reverse-transcribe cDNA Pool PCR Cell label Universal primer Oligo(dT) Second strand RNA synthesis Hybridize oligo High-throughput single-cell labeling (Hi-SCL) Hi-SCL Barcoded mRNA from single cells AA(A) n Single cell Cell suspension Each droplet with unique oligos Insert oligos in droplets Load single cells into droplets with lysis buffer Fuse droplets Pool all droplets cDNA synthesis and amplification Sequence Universal primer Oligo(dT) RT buffer High-throughput single-cell labeling with indexing droplets (inDrop) inDrop Barcoded mRNA from single cells AA(A) n Single cell Cell suspension Each microsphere with unique oligos Oligos attached to hydrogel Load single cells into droplets with lysis buffer Combine micro- spheres and droplets Pool all droplets UV primer release cDNA synthesis and amplification Sequence Photocleavable linker Oligo(dT) RT buffer Cell label A single nucleus RNA-Seq method (Nuc-Seq) Nuc-Seq AA(A) n Single cell Tissue Fixation and freeze Lyse and centrifuge Sort nuclei Nuclei mRNA fragment AAAAAA cDNA synthesis Tagmentation PCR AAAAAA TTTTTT CCC GGG Locked nucleic acid (LNA) CCC GGG Enrichment-ready fragment P5 P7 Index 1 Index 2 Gap repair and PCR Single-cell RNA barcoding and sequencing (SCRB-Seq) Div-Seq AA(A) n Single cell Tissue in vivo labeled with 5-ethynyl-2’-de- oxyuridine (EdU) Nuclei isolation Click-IT tagging FACS sort mRNA fragment AAAAAA cDNA synthesis Tagmentation PCR AAAAAA TTTTTT CCC GGG Locked nucleic acid (LNA) CCC GGG Enrichment-ready fragment P5 P7 Index 1 Index 2 Gap repair and PCR C H 3 O CH 3 Display methods on mobile device N N N N O O OH NH HN H H S Biotin Preparation of acylated RNA for biotin–streptavidin purification. DIBO, dibenzocyclooctyne N O N 3 O RNA N O O RNA N N N Biotin N N N O N 3 RNA + Acylation DIBO-biotin “click” 5-Ethynyl-2'-deoxyuridine (EdU) NH N HO HO O O O 5-iodouridine (5IU) N OH O OH OH NH O I O 4-thiouridine (4SU) N HO O OH OH NH O S 5-bromo uridine (5BrU) N OH O OH OH NH O Br O 6-Thioguanosine (6SG) N OH O OH OH N NH 2 NH N S Photoactivatable Nucleosides Locked nucleic acid (LNA) N OH O OH O N NH 2 NH N O N NH 2 N O N NH 2 N O CH 3 Cytosine 5-Methyl Cytosine N NH 2 N O CH 3 5-Methyl Cytosine N N O O Uracil Bisulfite conversion N 6 -Methyladenosine (m 6 A) N O O O O O OH OH N N N N CH 3 H P O O OH NH HN H H S Biotin Biotin-4-fluorescein (B4F) O O NH HN H H S O O O HO HO H N NH O p-benzoylphenylalanine (Bpa) O HO O H 2 N SUPeR-seq Single-cell universal poly(A)-indepen- dent RNA sequencing (SUPeR-seq) AA(A) n AAAAA Add poly(A) primer with T7 promoter and PCR target Reverse transcription and primer digestion with ExoSAP-IT PCR amplification Purification DNA AAAAA NNNNNT T T T T NNNNNT 15 NNNNNT 15 NNNNNT 15 AAAAA TTTTT AAAAA TTTTT PolyA RNA RNA cDNA RT primer with well barcode and UMI Transposase contains well-specific barcodes AA(A) n AAAA RNA AAAA TTTT TTTT Pool & sequence Single-cell combinatorial indexing-based co-assay that jointly profiles chromatin accessibility & mRNA (sci-CAR) Nuclei are indexed for ATAC-Seq and RNA-Seq using well-specific barcodes Addition of the 2 nd well barcodes tags each nuclei with two different combination of well barcodes, acting as a marker to contrast DNA fragments that are from different 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 Split Add RNA-Seq index Add ATAC-Seq index Nuclei suspension 25 nuclei/well gDNA in-situ tagmentation DNA with ATAC-Seq index DNA with RNA-Seq index Seq adapters 2 nd 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 β Very rare mutation Duplex sequencing detects rare mutations by sequencing and aligning both strands of the DNA P5 P7 P5 P7 A mutation occurs on both strands 12 random base index 12 random base index True variant Random error Ligate and PCR Rare variant Sequence Create single strand consensus sequence from every unique molecular tag Consensus Create 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 Sequence Oligonucleotide-selective sequencing (OS-Seq) captures and sequence gene targets on the flow cell Create target-specific oligos Extend and Denature Extend and Denature Extend and Denature Sequence reads 1 and 2 Fragment and add single adapters Genome DNA and mRNA sequencing (DR-Seq) DR-Seq AA(A) n Single cell polyA RNA DNA AA(A) n polyA RNA DNA Single cell RT with barcoded primer Lyse cell Ad-2 primer Split samples Quasilinear amplification Sequence gDNA amplification cDNA amplification TTTTTTTTTT AAAAAAA 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 Single cell polyA RNA DNA AA(A) n polyA RNA DNA Cell suspension Isolate single cell Separate the DNA and the RNA Lyse cell Sequence TTTTTTTTTT AAAAAAA Streptavidin magnetic bead with mRNA capture primer Streptavidin magnetic bead with mRNA capture primer TTTTTTTTTT AAAAAAA On-bead transcriptome amplification with Smart-Seq2 Whole-genome amplification with scBS-seq Very rare mutation Safe-SeqS DNA Shear Mutation Amplify and solid phase capture Sequence Safe-sequencing system is a unique molecular identifier (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 DNA DNA-protein complex DNA extraction Crosslink proteins and DNA Sample fragmentation Single cell chromatin immunoprecipitation (scChIP-seq) Single-cell triple omics sequencing (scTrio-seq) scTrio-Seq AA(A) n Single cell polyA RNA DNA DNA methylation Cell suspension Isolate single cell Lyse and centrifuge Supernatant Nucleus AA(A) n polyA RNA Add carrier RNA AA(A) n T T (T) n cDNA synthesis PCR and sequence Add poly A with TDT Hybridize oligo AA(A) n DNA Add sequencing adapters PCR and sequence Align sequences Methylated regions Methylated adapter End repair and ligation Bisulfite conversion Converted fragments MspI digestion PCR and sequence Methylated DNA scAba-Seq DNA Detect 5hmC marks in single cells with AbaSI nuclease (scAba-seq) Glucosylated 5-hmC 5hmc residues T4-βGT Hydroxy-methyl- ated DNA AbaSI Ligate Pool T7 amplification Primer Illumina 5’ adapter T7 promoter Adapter with cell-specific barcode Single cell Droplet-based single-cell ChIP-seq (Drop-ChIP) Drop-ChIP Single cell Barcoded sequences from single cells Cell suspension Droplet with unique oligos Load single cells into droplets with lysis buffer and MNase Fuse droplets Pool all droplets Sequence Chromatin immuno- precipitation Single cell scATAC-Seq (Microfluidics) Fragmented and primed DNA Single-cell assay for transposase accessible chromatin (scATAC-Seq) Lyse and introduce Tn5 transposase Pool libraries from all cells Amplify with cell-specific barcodes Insert in regions of open chromatin Cell suspension Microfluidics 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 amplification Create sequencing library Sequence capture Nucleus Single cell scATAC-Seq (Cell index) DNA Single-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 SMDB Single-molecule droplet barcoding (SMDB) DNA templates Single template encapsulation Template amplification Template fragmentation Barcode every droplet Pool for library prep DNA gRNA efficiency i5 i7 UMI gRNA UMI-based pooled CRIPSR screening for single-cell lineage tracing and quantification of gRNA efficiency (CRISPR-UMI) Each gRNA construct contains random 10 nt UMI barcode, gene-specific spacer sequence, and Illumina indexed adaptors. They are also flanked by PacI restriction sites Strong limiting dilution isolates different editing outcomes to its own colonies Each cell in a colony was edited by specific gRNA hence contains the same UMI sequence gRNA sequence is read and resulting data used to determine which gRNAs were most efficient in creating the desired outcome gRNA CRISPR/Cas9 Cell suspension Cell CRISPR-UMI gRNA design Edit, select, expand Expand Purify DNA and amplify gRNA constructs Limiting 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 amplification 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-PCR Cell-specific barcodes DNA with spatial info Frozen tissue slices UV-catapulting Cell-specific barcodes TSCS Single cell Random primer Methylated DNA Bisulfite conversion Amplify and sequence Single-nucleus methyl- cytosine sequencing (snmC-Seq) Isolated single cell Lyse Random priming Extend Pool samples and Adaptase reaction Adapter snmC-Seq Methylated DNA Single cell Isolate nuclei and nucleosome depletion Pool Bisulfite conversion, linear amplification, and PCR amplification 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-specific index and read 1 primer sequence This step converts unmethylated cytosines, tags fragments with a second well-specific barcode, and attaches sequencing adapters. Sequencing-ready cDNA 1st adapter Sequencing adapters + well-specific index Pool sci-MET Cell barcode T7 promoter Tn5 bind site Pool Pool & sequence Single nuclei Single-cell transposome hyper-sensitive site sequencing (scTHS-Seq) Split Split In-vitro transcription amplification 3’ end transposition & end fill-in Sequencing adaptor ligation & amplify Nuclei suspension Open DNA 2000 nuclei/well gDNA 100 nuclei/well 3’ adaptors Illumina indexed adaptors gDNA 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 Split Fixed cells Well-plate Protein-DNA complex 1st barcode 2nd barcode Pool Pool & purify Split 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 Single cell polyA RNA DNA AA(A) n polyA RNA DNA Cell suspension Isolate single cell Separate the DNA and the RNA Lyse cell Sequence TTTTTTTTTT AAAAAAA Streptavidin magnetic bead with mRNA capture primer TTTTTTTTTT AAAAAAA On-bead transcriptome amplification with Smart-Seq2 Whole-genome amplification with MDA AA(A) n polyA RNA Copy Number Alterations DNA methylation scBS-Seq Supernatant Nucleus AA(A) n polyA RNA Add carrier RNA AA(A) n T T (T) n cDNA synthesis PCR and sequence Add poly A with TDT Hybridize oligo AA(A) n DNA Add sequencing adaptors PCR and sequence Cell suspension 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 Bisulfite conversion Random primer 2 Align fragments from every UMI and sequence First random priming Second random priming Repeat 4 times PCR Extend Adapter Adapter Exo I and purify Single cell scTrio-Seq2 CpG dinucleotides Methylated CpG Single-cell nucleosome, methylation and transcription sequencing (scNMT-Seq) Isolate single cell Lyse and GpC methylase labelling AA(A) n Single cell polyA RNA DNA methylation Chromatin accessibility Transcriptome DNA methylation Chromatin accessibility mRNA DNA TTTTTTTTTT AAAAAAA Streptavidin magnetic bead with mRNA capture primer Methylated CpG indicates accessible DNA Isolate mRNA TTTTTTTTTT AAAAAAA On-bead transcriptome amplification with Smart-seq2 Whole genome bisulfite sequencing with scBS-seq scNMT-seq Biotin 6 rounds of random priming with biotinylated adapters Methylated GpC dinucleotides mark the absence of nucleosomes Capture first strand on Streptavi- din-coated magnetic beads Streptavidin 2nd random primer Amplify and sequence Methylated CpG dinucleotides Methylated GpC dinucleotides GpC methyl- transferase Lyse Chromatin NOMe-Seq PBAT Bisulfite conversion Chromatin overall omic-scale landscape sequencing (scCOOL-Seq) Single cell DNA methylation CNV and ploidy Chromatin accessibility and nucleosome position scCool-Seq Sort cells to microplate wells Hypotonic lysis Physical separation Supernatant Library prep Nuclei Total RNA From cellular mRNA From genomic DNA Antibody Surface antigen Microwell Magnetic microbead DNA 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) n Non-polyA RNA PolyA RNA SIDR Single cell Crosslinked chromatin dsDNA Tagmented DNA Hi-C Restriction digest Single cell Fixed cell Single-cell chromatin conformation capture method with multiplex end-tagging amplification (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 efficiency 20 different 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 artificial chimeras Sophisticated algorithm can also identify chromosomal haplotypes linked by contact META transposome Transposase Seq adapters META PCR primers META barcodes DNA META PCR primer 20 bp META barcode DNA ligase Lyse cell Amplify with META primers Dip-C 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 • [email protected] • illumina.com Illumina, 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 Scientific Affairs. 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 Scientific Affairs with any questions, comments, or suggestions. References Act-Seq Wu Y. E. et al. (2017) Neuron 96(2): 313-329 CEL-Seq Hashimshony T. et al. (2012) Cell Rep 2: 666-673 CirSeq Acevedo A. et al. (2014) Nature 505: 686-690 CITE-Seq Stoeckius M., et al. (2017) Nat Methods 14(9): 865-868 CLaP Binan L. et al. (2016) Nat Commun 7: 11636 CRISPR-UMI Michlits G. et al. (2017) Nat Methods 14(12): 1191-1197 CROP-Seq Datlinger P. et al. (2017) Nat Methods 14(3): 297-301 CytoSeq Fan H. C. et al. (2015) Science 347: 1258367 Digital RNA Shiroguchi K. et al. 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(2018) Elife 7: e32942 Sequencing by Synthesis TruSeq ™ PCR Free Double-stranded DNA Fractionate Size select A-overhang End repair Phosphorylate P P A A P P T P P5 P7 Index T P P5 P7 Index Adapter ligation P5 P5 P7 Index P7 Index Add Adapters Product ready for cluster generation TruSeq ™ Nano Double-stranded DNA Fractionate Size select A-overhang End repair Phosphorylate P P A A P P T P P5 P7 Index 1 Index 2 Index 2 Index 1 T P P5 P7 Adapter ligation Denature and amplify Add Adapters P5 P7 Index 1 Index 2 Index 2 Index 1 P5 P7 P5 P7 Index 1 Index 2 Double-stranded DNA Product ready for cluster generation TruSeq ™ Small RNA 3’ 5’ Small RNA fragment Ligate adapters Add primer Reverse transcription Denature and amplify 5’ Adapter 3’ Adapter P7 Index 1 P5 P5 P7 Index Product ready for cluster generation A A T T C G C A A T T C G C A A T T C G C A A T T C G C A A T T C G C Synthesize second strand The second read is sequenced Sequence Index2 A A T T C G C Deblock P5 primer and add unlabeled bases Read 2 primer The forward- strand is cleaved and washed away A A T T C G C A A T T C G C A A T T C G C A A T T C G C A A T T C G C A A T T C G C 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 Fold over and hybridize to first primer Sequence Index1 Index 1 primer The read product is washed away TruSeq ™ RNA Exome Elute Target Target P5 P7 Index 1 Index 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 Expression Target ULSO DLSO Total RNA cDNA Hybridization P7 Index 1 P5 P5 P7 5’ P 5’ P Index 2 Target Index 1 Index 2 Product ready for cluster generation Add custom primers Denature and amplify Extension-Ligation Nextera ™ Library Preparation Transposase DNA ~300bp Tagmentation Amplification P5 P7 Index 1 Index 2 P5 Index 2 Index 1 P7 Product ready for cluster generation Nextera ™ Mate Pair Adapter ligation Isolate biotinylated fragment Denature and amplify P5 P7 Transposase DNA Tagmentation Circularize R R R R Biotinylated junction adapter R R R R R R Fragment R R R R P5 P7 P5 P7 R R Product ready for cluster generation Nextera ™ Rapid Capture Elute Target Target P5 P7 Index 1 Index 2 Product ready for cluster generation Denatured and pooled fragments from Nextera library Capture on streptavidin magnetic beads Hybridize probes to targets Biotinylated target probe AmpliSeq ™ for Illumina Remove primer sequences Add sequencing primers Index 2 Index 1 P5 P7 PCR Index 1 P7 Index 2 P5 Product ready for cluster generation DNA/cDNA DNA RNA 5’ 3’ PCR Reverse transcribe Ligate adaptors TruSeq ™ RNA Total RNA TTTTT AAAAA polyA select Fragment Random hexamer First and second strand synthesis 5’ 3’ TruSeq ™ Stranded RNA RNA/mRNA Random primer Create cDNA Create second strand cDNA End repair Phosphorylate A-overhang Adaptor ligation Denature and amplify cDNA dUTP + dCTP + dATP + dGTP dTTP + dCTP + dATP + dGTP P5 P7 P5 P7 Index 1 Index 2 U UU U U U U U U U U Sense strand A A P P U UU U U U U U U U Sense strand P7 Index 1 P5 Index 2 U UU U U U U U U U Index 2 P5 Index 1 P7 Sense strand P5 P7 U UU U U U U U U U Sense strand Block polymerase Product ready for cluster generation 5’ 3’ AAAAA SureCell ™ WTA 3’ Amplification 3’ enrichment and sample indexing Product ready for cluster generation Direct cDNA Nextera tagmentation Cell lysis mRNA hybridization Single cells encapsu- lated in droplets cDNA synthesis and barcoding Barcoded beads UMI Barcode Read1 TTTTT AAAAA mRNA UMI Barcode Read1 TTTTT mRNA AAAAA UMI Barcode Read1 TTTTT mRNA AAAAA Index P7 P5 Index P7 P5 UMI Barcode Read1 cDNA
