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DNA
DNA Rearrangements and Markers DNA-Protein Interactions
Bisulfite conversion
Genome
NH2 NH2 Single cell RNA RNA On-bead transcriptome H2 N NH2 ChIP-Seq
RAD Restriction sites AA(A)n AA(A)n AAAAAAA
amplification with Smart-seq2
ChIP-exo
CH3
scM&T-seq
AAAAAAA TTTTTTTTTT N
N N TTTTTTTTTT
PE RAD-Seq DNA DNA Whole genome bisulfite Me
Restriction-site associated DNA marker Restriction Add barcoded adapters Shear and Add P2 Amplify DNA O N O N sequencing with scBS-seq HT-ChIP Chromatin immune precipitation (ChIP-Seq), DNA-protein complex Crosslink proteins and DNA Sample fragmentation Exonuclease digestion Immunoprecipitate DNA DNA
generation (RAD) digestion size select adapter O
Cytosine 5-Methyl Cytosine
Methylome and transcrip-
tome sequencing from a
Cell
suspension
Isolate single
cell
Lyse cell Streptavidin magnetic bead
with mRNA capture primer
Separate the DNA and the RNA Sequence Align RNA and
methylome O Mint-ChIP High-throughput chromatin immunoprecipi-
tation (HT-ChIP))
extraction
single cell (scM&T-seq) O- O
NH
Genome Restriction sites
ddRADseq Single cell RNA
N
Fe
O-
RNA On-bead transcriptome O-
AA(A)n AA(A)n AAAAAAA
amplification with Smart-seq2
N DNase-Seq
Double digest restriction-site associated Restriction Add barcoded adapters Second restriction digest Add P2 Amplify
scMT-seq
NH2 TTTTTTTTTT
DNA O AAAAAAA
TTTTTTTTTT
DNA marker generation (ddRADseq) digestion adapter
N N
CH3 DNA DNA Whole genome amplification N
H
O- DNaseI-Seq
with RRBS DNase I hypersensitive sites sequencing Active chromatin DNase I digestion Isolate trimmed complexes DNA extraction DNA
Methylome and transcrip- Cell Isolate single Lyse cell Streptavidin magnetic bead Separate the DNA and the RNA Sequence Align RNA and O (DNase-Seq, DNaseI-Seq)
Genome BsaXI restriction sites N N
O O Methidiumpro-
2b-RAD Uracil 5-Methyl Cytosine
tome sequencing from a
single cell (scMT-seq)
suspension cell with mRNA capture primer methylome
pyl-EDTA (MPE)
Supernatant Nucleus
Restriction-site associated DNA marker
generation (RAD) with type IIB restriction
Restriction digestion with type IIB
restriction endonucleases e.g. BsaXI
Add restriction-
site-specific adapters
Amplify Add sample-specific
adapters
Amplify DNA
Single cell
RNA
AA(A)n AA(A)n
DNaseI-SIM
RNA T T (T)n AA(A)n
endonucleases (2b-RAD) AA(A)n
scTrio-seq DNA
Supernatant
DNA
Add carrier RNA Hybridize oligo cDNA synthesis Add poly A with TDT PCR and sequence
Nuclei
Genome Restriction sites
SLAF-seq DNA methylation Add sequencing adaptors PCR and sequence Dnase I simplified in-nucleus
method (Dnase I SIM) for plants
Cells Lyse and
centrifuge
Sort
nuclei
Nucleus DNase I digestion Terminate DNase I Polish ends DNA DNA
Nucleus digestion extraction
Methylated DNA
Specific locus amplified fragment sequenc- Digest with MseI Add MseI Digest with AluI PCR with barcoded Purify and Add sequencing PCR DNA
ing (SLAF-seq) for large scale genotyping adaptors MseI primer pool adaptors
Single-cell triple omics Cell Isolate Lyse and MspI Methylated Methylated End repair Bisulfite Converted PCR and sequence Align sequences
sequencing (scTrio-seq) suspension single
cell
centrifuge digestion regions adapter and ligation conversion fragments MAINE-Seq
Genome High quality DNA DNA to be captured MNase-Seq
hyRAD scBS-seq MNase-assisted isolation of nucleosomes (MAINE-Seq).
N
N Random primer 1
Adaptor Random primer 2
Adaptor
Nucleo-Seq Also Micrococcal nuclease sequencing (MNase-Seq)
Open chromatin MNase digestion Isolate trimmed complexes DNA extraction DNA
Hybridization RAD (hyRAD) Prepare RAD-seq Adaptor removal Biotinylated Shotgun library Hybridize to probes Streptavidin DNA N N
for degraded DNA library and size select and labeling probes pull down Single-cell bisulfite Isolated Lyse Methylated DNA Bisulfite First random Repeat Extend Exo I and Second random PCR Align fragments from every Sequence
sequencing (scBSBS-seq) single cell conversion priming 4 times purify priming unique molecular tag
Plate 1 Plate 2
Genome Restriction sites
Rapture
Well 1
Well 2
H3C Single cell 5hmc residues FiT-seq
scAba-seq
CH3 Adaptor with cell-specific barcode
O Illumina 5’ adaptor Fixed-tissue chromatin immuno- FFPE-fixed Deparaffination Heat 40°C in SDS Proteinase K Enzyme Sonicate Soluble Immunoprecipitate Cromatin Purification DNA
Restriction-site associated Streptavidin T7 promoter precipitation sequencing (FiT-Seq) chromatin and rehydration digestion inactivation extract elution
Digest with Add well-specific Pool wells Restriction Library prep with Pool Hybridize Biotinylated Streptavidin DNA
DNA marker generation restriction barcodes and shear pull down enzyme digest plate barcodes capture bait pull down Detect 5hmC marks in single cells Hydroxy-methyl- T4-βGT Glucosylated 5-hmC AbaSI Primer Ligate Pool T7 amplification DNA
(RAD) capture (Rapture) enzyme with AbaSI nuclease (scAba-seq) ated DNA
Display methods on O
mobile device
Dige- Genome Cas9 target Deletion
Novel retrotrans- Retrotransposon N
Wild type DNA + N
PAT-ChIP
nome-seq Insert
scRC-Seq
position events binding site
N Pathology tissue chromatin FFPE-fixed Deparaffination MNase digestion Sonicate Soluble Immunoprecipitate Reverse crosslink and Purification DNA
In vitro Cas9-digested whole-ge- Target site digestion Sequence Align and determine immunoprecipitation (PAT-ChIP) chromatin and rehydration extract and chromatin elution proteinase K digestion
nome sequencing (Digenome-seq) sequence breaks N3
Single cell retrotransposon Genomic DNA Cell FACS Pick Nucleus Whole genome Create sequencing Sequence capture Enriched library Acylation
Ribonucleotides capture sequencing (scRC-Seq) suspension isolation nuclei amplification library
Genome 2’,3’-cyclic phosphate
HydEn-seq 5’
3’
R R
3’
5’ 5’HO 5’P-O
R R O
DNA
Single cell
Hydrolytic end sequencing (HydEn-seq) to reveal replicase- and Alkaline Phosphorylation (T4 PNK Ligate oligo with 5’-amino- Add sequencing primers, Map locations
scATAC-Seq O X-ChIP
strand-specific patterns of ribonucleotides in the genome hydrolysis (KOH) 3-phosphatase minus) terminated C6 spacer PCR and sequence on the genome High resolution of mapping of in vivo Crosslink Lyse cells Cross-linked chromatin MNase digestion Sonicate Soluble Immunoprecipitate DNA
NH2 (Cell index) N
chromatin associated proteins (X-ChIP) cells in vivo extract and DNA extraction
R Single-cell assay for transposase Cell suspension Isolate Nuclei Split Barcode each well Pool and Split sample PCR-barcode Pool for library DNA N3
Genome with ribonucleotides R mA R Am R N accessible chromatin (scATAC-Seq) sample with Tn5 transposase dilute every well prep
T
2’P
T
R R R R R DIBO-biotin “click”
Ribose-seq
T
2’P
A Am A Am A R
5’ P 5’ P A
T
T A Am
T
ORGANIC
A T
P 5’ A P 5’ mA A T mA A T mA A T N O Microfluidics device
Detect ribonucleotides Fragmented dA tailing Adaptor ligation Alkali treatment Self-ligation by Degradation of Remove 2’-phos- DNA for Single cell O Occupied regions of genomes from Cell Isolate nucei Isolated chromatin Soluble extract Immunoprecipitate
DNA
MNase digestion DNA
embedded in DNA genomic DNA AtRNL linear ssDNA phate and PCR sequencing R affinity-purified naturally isolated and DNA extraction
(Ribose-seq) Cytosine scATAC-Seq O chromatin (ORGANIC)
Sequencing Primer NH2 (Microfluidics) Single-cell assay for transposase Cell Isolate Lyse and introduce Insert in regions of open chromatin Fragmented and primed Amplify with Pool libraries DNA N
CH3 accessible chromatin (scATAC-Seq) suspension single cell Tn5 transposase cell-specific from all cells N
Genome Potential G-quadruplex K+ or PDS Polymerase stalls
N barcodes N N
G4-seq ATAC-Seq
N O
Single cell Assay for transposase accessible Open DNA Tn5 Transposome Insert in regions of open chromatin Fragmented and primed DNA purification DNA
Droplet with
Determine the location of Target sequence Fragment and Hibridize Read reference Denature and remove Hibridize Add K+ or PDS to Read stabilized Compare
5-Methyl Cytosine
R Drop-ChIP unique oligos
chromatin (ATAC-Seq) Amplification
potential G-quadruplexes in create library on sequencing sequence read fragment sequencing stabilize G4 regions squence sequence reads
DNA (G4-seq) flow cell primer primer 1 and 2 scChIP-seq Biotin Read primer
NH2 T7 promoter
Droplet-based single-cell Cell Load single cells into droplets Fuse droplets Pool all droplets Chromatin immuno- Sequence Barcoded sequences Preparation of acylated THS-seq
XC
CX
X CXXC CXXC CXXC ChIP-seq (Drop-ChIP) suspension with lysis buffer and MNase precipitation from single cells
XC
CpG island HO N
CX
Reference sequence
N O HELP-Seq Bisulfite conversion of genomic DNA without Methylated DNA Bisulfite conversion T T T C G Converted single-stranded Random priming 3’ tagging PCR DNA
RC-Seq: Retrotransposon Genomic DNA Fractionate DNA Hybridize Microarray with transposon
Read2
Transposon sites
Align
Novel retrotransposition
events R
shearing. HpaII tiny fragment enrichment by
ligation-mediated PCR (HELP-Seq)
fragments DNA synthesis FAIRE-seq
capture sequencing binding sites
fragments
5-carboxylcytosine (5caC)
Streptavidin
Sono-Seq Formaldehyde-assisted isolation of regula- Crosslink protein and DNA with formalin Sonicate Phenol extract and purify DNA
Open DNA DNA
Biotin
Random primer 2 tory elements (FAIRE-Seq) and sonication of from the aquous phase
Transposon Transposon 20bp MmeI Adaptor cross-linked chromatin (Sono-Seq)
Adaptor
TN-Seq MmeI 20bp PBAT
Post-bisulfite adaptor Methylated DNA Bisulfite First random Capture first strand on Streptavi- Second random Generate second strand Elution DNA with adaptors
INSeq Transposon sequencing (TN-Seq) Inverted MmeI MmeI Transposon Protected Protected
recognition site recognition site MmeI digestion Add adapters PCR and sequence
tagging (PBAT) conversion priming din coated magnetic beads priming CpG dinucleotides CpG dinucleotides
and insertion sequencing (INSeq) insertion sites
NOMe-Seq Methylated CpG Methylated CpG
Control
Protected
methylated
Unprotected
methylated
MmeI AluI MmeI
Nucleosome Occupancy Methylome- Open DNA GpC methyltransferase (M.CviPI) and Bisulfite conver- M.CviPI
+AID H1P1 P2 H2 Sequencing (NOMe-Seq), a single-molecule S-Adenosyl methionine (SAM) Protected Unprotected
AID-dependent rearrangement A A sion BS-Seq
TC-Seq
I-SceI site BSPP nucleosome positioning assay unmethylated unmethylated
-AID A A Bisulfite sequencing with CpG island Bisulfite Bisufite-converted Padlock Hybridize Extension Exonuclease PCR End repair DNA with
padlock probes (BSPP) conversion DNA probe and ligation digestion and adaptor adaptors
TC-Seq: translocation
capture sequencing
Genomic DNA Infect I-Sel Sonicate, blunt
and A-tail
Ligate linkers
Cut I-Scel
Purification Semi-nested PCR
Linker cleavage
DNA ligation Histone
methylation
Ig-seq CDR3 junction region V D J Constant 8N UID O
RRBS
H2N
ChIP-Seq of methylated histones DNA-protein complex Crosslink proteins Sample Exonuclease digestion Immunoprecipitate DNA extraction DNA
Rep-Seq 8N UID (Histone methylation) and DNA fragmentation
DNA sequencing of immunoglobulin genes Extracted RNA Reverse transcription Second strand synthesis PCR Purify DNA H2N H2N scRRBS Reduced representation bisulfite Methylated MspI Methylated Methylated End repair Bisulfite Converted fragments PCR DNA
MAF
O O
(Ig-seq), repertoire sequencing (Rep-Seq), and N sequencing (RRBS-Seq). DNA digestion regions adapter and ligation conversion HO OH
molecular amplification fingerprinting (MAF) NH NH Single cell RRBS (scRRBS) HO O
O O
Single-strand break HO
O
O- O-
NH
ChIPmenta-
SSB-Seq tion
N O
BSAS O P O P O O
Chromatin immunoprecipitation Chromatin Chromatin immunoprecipitation Tn5 Transposome Adaptor insertion Fragmented DNA purification DNA
Map DNA single-strand Genome DNA DNA Pol I, dU-digoxigenin, and dNTPs DNA isolation and random shearing Immunoprecipitate with DNA Bisulfite amplicon Bisulfite Bisufite-con- PCR with primers specific for Amplicons Transposome with
OH OH
with sequencing library preparation and primed Amplification
Methylated DNA Tagmentation DNA
breaks (SSB-Seq) anti-digoxigenin antibody Pyridostatin (PDS) sequencing (BSAS) conversion verted DNA bisulfite converted DNA adaptor HO HO by Tn5 transposase (ChIPmentation)
Uridine diphosphate
glucose (UDP-Glu)
MspI
Double-strand break TT TT
TT C C G G C C G G Chia-PET
TTTT
TTTT
TTTT
TT
BLESS
C C G G C C G G
Methyl-Seq
TT
TT
TT
TTTT
TT TT TT TT
TTTT
TTTT
TTTT
TT TT TT
HpaII
MRE-Seq
C C G G C C G G
Breaks labeling and enrichment on Genome DNA Biotinylated Ligate primer DNA isolation and Capture on streptavidin Distal Digest with DNA Methyl-seq and MRE-Seq use C C G G C C G G Chromatin interaction analysis by Sample fragmentation Immunoprecipitate Ligation Restriction enzyme digestion DNA
streptavidin and sequencing (BLESS) proximal primer random shearing beads primer I-SceI and PCR methyl-sensitive enzymes to Methylated sites Split sample Restriction Sequence Align sequences and Identified paired-end tag sequencing (ChIA-PET)
identify methylation patterns in the genome enzyme digest determine undigested sites methylation site
Excision circle sequencing Lymphocyte-specific recombination activat- Synapsis, cleavage and coding Resolved genomic Liberated excision circle with Align read pairs to
(EC-seq) ing gene (RAG) recombinase, bound genome end hairpin formation coding junction resolved signal junction reference genome 5hmc residues
4-C
O
O
JBP1-seq OH
HO
CH3 J-binding protein 1 sequencing Hydroxy-methyl- Transposomes Tagmentation T4-βGT Glucosylated 5-hmC JBP1-magnetic bead PCR DNA HO
O
Bubble-Seq CH3
(JBP1-seq), for genome-wide profiling
of 5-hydroxy-methylcytosine (5hmC)
ated DNA pull down HO
OH
O NH2 Chromatin conformation
capture circular (4-C)
Crosslink proteins and DNA Sample fragmentation Ligation Restriction digest Self-circularization
and Reverse PCR
DNA
OH N
O O O
Libraries of restriction fragments Bubble-containing Restriction Cast fragments in Run gel Recover bubble-con- DNA extraction Add sequencing DNA 5hmc residues N O US DS
that contain replication initiation fragment digest trapping gel taining plug primers
Aba-seq
HN Universal primer
Adaptor primer primer
UMI-4C
O O
sites (bubbles) in vivo R
N HN Pad
H AbaSI coupled with sequencing Hydroxy-methyl- T4-βGT Glucosylated 5-hmC AbaSI Biotinylated Ligate Fragment Streptavidin magnetic DNA Glucosylated 5hmc
Biotin (Aba-seq) to map high-resolution ated DNA primers bead pull down
O- O- O- O N O
gDNA gDNA O- O- O- O hydroxymethylome (5hmC) Targeted chromosome conformation Crosslink proteins HC DpnII Ligation Reverse crosslink and Sonicate Single end Nested PCR amplification DNA
NSCR RNA
primer SNS SNS O O O O
5mC 5hmC 5mC g5hmC 5cmC g5hmC N3 N3S-S Biotin
capture (4C) with unique molecular
identifiers (UMI-4C)
and DNA digestion proteinase K digestion adaptor ligation
g5hmC
5mc residue βGT TET g5hmC
Nascent strand capture and DNA replication bubble Denaturation and 5’-biotinylation Streptavidin pull-down RNase I Isolate and DNA
release (NSCR) size-selection digestion amplify SNS Digoxigenin TAmC-Seq C T C C G C T C C G C T C C G C T C C G C T C C G
T7 T3
Tet-assisted 5-methylcytosine βGT-catalyzed Biotinylation Streptavidin pulldown
sequencing (TAmC-Seq)
Glucosylation Oxidation
UDP-6-N3 glucosylation and DTT cleavage
DNA
5-C
BrdU
Nascent DNA
Repli Seq 5fc residue
C mC
5m 5h 5fc 5ca
C Blocked 5hmC residue 5hmC residue N3 N3S-S Biotin
Chromatin conformation capture Crosslink proteins and DNA Sample fragmentation Ligation LMA: Ligation-mediated amplification DNA
fC-Seal C C C C C C C C C C C C C C C C C C C C C C C C C carbon copy (5-C)
Repli Seq—to map temporally ordered Run-on with analog Sort cells and lyse Bead coated with Elute Purify DNA A 5-formylcytosine-selective βGT-catalyzed NaBH4 Convert 5fc to βGT-catalyzed Biotinylation Streptavidin pulldown DNA
replicating DNA anti-BrdU antibody End Repair chemical labeling (fC-Seal) approach glucosylation 5hmc UDP-6-N3 glucosylation and DTT cleavage
for genome-wide profiling of 5fC PB-seq
C mC C
5m 5h 5fc 5ca
Biotin
Nascent DNA 5fc residue N3 N 3S-S N 3SH Protein/DNA binding (PB–seq), to DNA-protein complex Purify DNA Shear DNA Hybridize with Isolate protein-bound DNA DNA
NS-seq fC-CET C C C C C C C C C C C C C C C C C C C C C C C T C determine the binding energy landscape DNA-binding protein DNA extraction
5fC based on selective chemical Azido 1,3-indandione DBCO-S-S-PEG3-biotin Pulldown, Adaptor ligation Purify DNA
Pu-seq Origin of replication Index 2 Index 1
labeling of 5fC and subsequent (AI) NaOH and DTT and PCR 5’ 3’
Nascent strand sequencing (NS-seq) to discover Digestion Purify C-to-T transition during PCR 3’ R 5’ U U U
DNA replication origin Lambda 5´ to 3´ exodeoxyribonuclease (λ-exo) Amplify DNA
DNA replication origins and G4 structures Polymerase usage sequencing Alkali Klenow reaction(+ random Attach Uracil DNA glycosylase- PCR and DNA
C mC C
5m 5h 5fc 5ca (Pu-seq) treatment primer, dATP, dGTP,dCTP, dUTP) adaptors and DNA lyase (USER) purify
o-acylisourea Control C C C C C T C C T T
C mC C C mC C C mC
DNA Low-Level Detection True variant AI-mediated cyclization CAB-Seq 5caC residue C
5m 5h 5fc 5ca
C C C C C
5m 5h 5fc 5ca
C C C C
Nu S-S
C
5m 5h 5fc 5caC S-S
C C C C
Biotin
C C C C C
5caC S
T C C T C Break-seq
Breakage at replication fork
5’
3’
Break 3’
5’
5’
3’
3’
5’
Index 2 Index 1
Read1 Sample index labeling in fC-CET Chemical modification-assisted 1-ethyl-3-[3-dimethylamino-propyl]-car- Linker Streptavidin pulldown Bisulfite treatment Sequence
Degenerate Double-stranded break DNA trapped in End-repair with dGTP, dCTP, Elution and Streptavidin magnetic Attach PCR and DNA
Gene O bisulfite sequencing (CAB-Seq) bodiimide hydrochloride (EDC) chemistry and DTT cleavage PCR amplification labeling to map chromosome agarose gel dTTP, and biotinylated-dATP fragmentation bead pull down adaptors purify
smMIP molecular tag
Read2
Random error
NH2 for 5caC detection O
NH H O breaks (Break-seq)
C mC C
Single Molecule Molecular Genomic DNA Copy target sequence Exonuclease PCR amplification Align fragments from every Corrected N 5m 5h 5fc 5ca Bisulfite treatment HN OH NNNN
Inversion Probes (smMIPs) for unique molecular tag sequence C mC C T C C T T NNNN
N 5hmc residue 5m 5h 5fc 5ca Control C C C C C PCR amplification Breakage in genome
LAM-HTGTS
detecting low frequency targets O H S
Degenerate DNA
oxBS-Seq C C C C C 5fc Bisulfite treatment T C T T T DNA Biotin
Short tandem repeat (STR) Strain I Strain I Oxidative bisulfite sequencing KRuO PCR amplification Linear amplification-mediated Fragmentation by LAM-PCR with Streptavidin magnetic Adaptor ligation Nested Enzyme Tagged PCR
MIPSTR molecular tag Purify DNA
4 C C C C C
(oxBS-Seq) to map 5-methylcytosine high-throughput genome-wide sonication biotinilated primer bead pull down PCR blocking
Strain II Strain I N3
5fC and 5-hydroxymethylcytosine
Targeted capture of STR Targeted STR Copy target STR Amplify and sequence Natural variation between individuals Somatic variation within an individual C mC C sequencing (LAM-HTGTS)
5m 5h 5fc 5ca
loci by smMIPs (MIPSTR) Bisulfite treatment
C mC C
5m 5h 5fc 5ca C C C C C T C C T T A
5fc residue Control PCR amplification Breakage in genome
A
A
RedBS-Seq mC HTGTS
A
C C C C C
5h
A
A
3’ blocked random hexamer primers Nascent replication fork Reduced bisulfite sequencing NaBH Bisulfite treatment DNA
MDA caMAB-seq
O
Genome O 4 C C C C C T C C C T Linear amplification-mediated Fragmentation by End repair 3’ A addition Adaptor ligation Streptavidin magnetic Nested Tagged PCR Purify DNA
(redBS-Seq), to map 5-formylcyto- PCR amplification
sine (5fC) in DNA high-throughput genome-wide sonication and PCR bead pull down PCR
IMS-MDA Multiple displacement amplification (MDA). Hybridize primers Phi 29 Synthesis Phi 29 Synthesis S1 nuclease Amplified DNA N3 C mC
5m 5h 5fc 5ca
C sequencing (HTGTS)
Immunomagnetic separation for targeted Bisulfite treatment
MIDAS bacterial enrichment for MDA (IMS-MDA) 5fc residue
C mC
5m 5h 5fc 5ca
C
Control
C C C C C
PCR amplification
T C C T T Forward sequencing adaptor
Sequencing primer pD40htSELEX
Fusion
Microwell displacement amplification Transcription factor binding site Barcode to identify sample
fCAB-Seq SELEX
Hybridize primers Synthesis C C C C C protein Luciferase
system (MIDAS) O Blocked 14N with all possible combinations DNA binding region
27-bp common sequence N
5fC chemically assisted bisulfite Bisulfite treatment DNA Reverse sequencing adaptor Luciferase
Genome
8 random nucleotides N sequencing (fCAB-seq) method for
O-ethylhydroxylamine (EtONH2) C C C C C
PCR amplification
T C C C T
SELEX-seq High-throughput systematic Target Ligand
Streptavidin binding peptide
Expression vector Fusion protein Matching ligand Wash and Recovered PCR and Binding
the base-resolution detection of 5fC
MALBAC Cycles of quasilinear
amplification
N O C mC
5m 5h 5fc 5ca
C HT-SELEX evolution of ligands by exponential
enrichment (HT-SELEX)
sequence immobilized in well binds elute matching ligand Sequence site
Partial amplicons C mC C Bisulfite treatment T C C T T N3
5m 5h 5fc 5ca
DNA
Multiple annealing and looping-based Hybridize primers Bst DNA Denature Looped full PCR DNA 5fC/5caC residues Control
C C C C C
PCR amplification
amplification cycles (MALBAC) polymerase amplicons 5fC-AI 5m
C O
MAB-seq
C C C C C HO
Template Bisulfite treatment O
Denature M.SssI methylase-assisted bisulfite M.SssI C C C C C C C C T T DNA HO
OH
NH2
Protein binding site Klenow
Single cell genome sequencing (MAB-seq) to map 5fC/5caC
PCR amplification
N
HiTS-FLIP Target sequence
Binding site
H
C mC C
Cell 1 S
5m 5h 5fc 5ca
nuc-seq Flowcell
O
H
N C mC C Bisulfite treatment T C C T T N O
5m 5h 5fc 5ca
Cell 2 N C C C C C
5fC/5caC residues Control PCR amplification High-throughput sequencing: Prepare sequencing libraries and Hybridize Synthesize second Add fluorescent- Hybridize Elute with increas- Scan flowcell
SNES Cell 3
O
5m
C fluorescent ligand interaction sequence first strand primer strand with unmodi- ly labeled ligand ing stringency
RRMAB-seq
C C C C C R
N Bisulfite treatment DNA N3-5GMC profiling (HiTS-FLIP) Remove second strand DNA fied nucleotides
Reduced representation M.SssI Digest Add methylated M.SssI C C C C C C C C T T
Single G2/M nucleus sequencing of Cell sorting from Lyse cell Nucleus Phi 29 Limited amplification Synthesis S1 nuclease DNA N N
methylase-assisted bisulfite PCR amplification
cells in S phase (nuc-seq). Single G2/M distribution with MspI adapters
sequencing (RRMAB-seq) DNA adenine
nucleus exome sequencing (SNES) DNA-protein interaction methyltransferase (DAM) Specific and non-targeted
C mC C C methylation DpnI DpnII PCR
mC 5ca 5g 5ca 5ca
Fusion protein
O
References
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TruSeq PCR Free TruSeq Nano TruSeq Custom Amplicon TruSeq RNA TruSeq Small RNA TruSeq RNA Stranded TruSeq RNA Access TruSeq Targeted RNA Expression Nextera Library Preparation Nextera Rapid Capture Nextera Mate Pair
Double-stranded DNA Double-stranded DNA 5’ 3’ RNA Target Transposase
Double-stranded DNA 5’ 3’ Small RNA fragment Target Total RNA Target
AAAAA mRNA Random primer Transposase Denatured and R R Biotinylated junction adapter R R
Fractionate Pool stranded pooled fragments DNA
Fractionate Ligate adaptors dT TP + dC TP + dATP + dGTP Create cDNA RNA-Seq libraries from Nextera library
Size select cDNA
Size select Region of interest cDNA DNA Tagmentation
5’ Adapter 3’ Adapter
R R
End repair
End repair AAAAA Create second Biotinylated R R
Phosphorylate TTTTT polyA select dUTP + dC TP + dATP + dGTP ULSO DLSO
Add custom
Phosphorylate Add primer strand cDNA Biotinylated 5’ P target probe
Sense strand U primers
P
P
P
Custom Custom U U U U U U U UU U
target probe R
P Probe 1 Probe 2 Fragment End repair R
A-overhang Add custom probes A Phosphorylate 5’ P
Hybridization
A-overhang P Tagmentation Hybridize probes Circularize
A Reverse transcription Sense strand A-overhang Hybridize probes to to targets R R
P
Random hexam- AU U U U U U U UU U P
targets
A P
P A er ~300bp
R R
Moleculo
Sequencing by Synthesis
~10kb Sheared genomic DNA
End repair
Sequence A A A A A
Read 2 A A A A A
Adapter ligation
Forward Reverse primer
A
T
T
A
T
T
A
T
T
A
T
T
A
T
T
primer A
T
T
A
T
T
A
T
T
A
T
T
A
T
T Generate clonal pools
Forward Reverse strand strand C
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
C
G
Amplify
Transposase
C C C C C C C C C C
strand strand
Index 1
A
A
T Tagmentation
primer A
A
T
T
C
G
T C
C
G
C
~600bp
P5 P5 P5 Add indices
Index 2 Index 2 Index 2
Adapter hybrid- Reverse Remove Fold over and Synthesize Thousands of molecules are The reverse With each cycle, four fluores- The read Sequence The read Fold over and Deblock P5 Sequence Synthesize The forward- The second Index 1 Index 1 Index 1
izes to flowcell strand forward hybridize to second strand amplified in parallel strand is cently tagged nucleotides product is Index1 product is hybridize to primer and Index2 second strand is read is P7 P7 P7
syntesis strand second primer Bridge amplification cleaved and compete for addition to the washed away washed away first primer add unlabeled strand cleaved and sequenced P5 P7
washed away growing chain. Only one is bases washed away P5 P7 Prepared fragments Pool and purify
P5 P7
incorporated based on the
sequence of the template.