TITLE: SHARE-seq V1
AUTHORS: Sai  Ma, Aviv Regev, Jason Buenrostro

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SHARE-seq for joint profiling of chromatin accessibility and gene expression at scale.</div></div>

[STEPS]
?. [Anneal oligo plates]
Dilute 120µl Round 1 linker oligo (1mM) with 11880µl STE buffer. Mix 90µl diluted Round 1 linker oligo with 10µl Round 1 oligo (100µM) in a 96 well PCR plate.
?. [Anneal oligo plates]
Dilute 120µl Round 2 linker oligo (1mM) with 9480µl STE buffer. Mix 88µl diluted Round 2 linker oligo with 12µl Round 2 oligo (100µM) in a 96 well PCR plate.
?. [Anneal oligo plates]
Dilute 144µl Round 3 linker oligo (1mM) with 9360µl STE buffer. Mix 86µl diluted Round 3 linker oligo with 14µl Round 3 oligo (100µM) in a 96 well PCR plate.
?. [Anneal oligo plates]
Anneal Round 1, Round 2, and Round 3 plates.  AB195ºC2 min2slow ramp, -1ºC/cycle, 1 min per cycle320ºC2 min44ºCForever5Total~1 h 26 min
AB195ºC2 min2slow ramp, -1ºC/cycle, 1 min per cycle320ºC2 min44ºCForever5Total~1 h 26 min
?. [Anneal oligo plates]
After annealing, check if there is significant water evaporation for the wells at the corners. If there is, add water to compensate.
?. [Anneal oligo plates]
Aliquot 10µl of annealed oligo to a new plate. Should be enough for 9×3 plates. Store the plates at -20ºC.
?. [Anneal adapter oligo]
Dilute Phosphorylated_Read2, Read1, Blocked_ME_Comp to 100µM with the IDTE buffer. Prepare transposition adapter and tagmentation adapter mix in PCR tubes.  AB1Transposition adapter Volume (µl)2100µM Phosphorylated_Read26.53100µM Read16.54100µM Blocked_ME_Comp1351M Tris pH 8.00.2665M NaCl0.267Total26.5  AB1Tagmentation adapterVolume (µl)2100µM Read1133100µM Blocked_ME_Comp1341M Tris pH 8.00.2655M NaCl0.266Total26.5
AB1Transposition adapter Volume (µl)2100µM Phosphorylated_Read26.53100µM Read16.54100µM Blocked_ME_Comp1351M Tris pH 8.00.2665M NaCl0.267Total26.5
AB1Tagmentation adapterVolume (µl)2100µM Read1133100µM Blocked_ME_Comp1341M Tris pH 8.00.2655M NaCl0.266Total26.5
?. [Anneal adapter oligo]
Anneal oligos in a thermal cycler.  AB185ºC2 min2slow ramp, -1ºC/cycle, 1 min per cycle320ºC2 min44ºCForever5Total~1 h 14 min
AB185ºC2 min2slow ramp, -1ºC/cycle, 1 min per cycle320ºC2 min44ºCForever5Total~1 h 14 min
?. [Anneal adapter oligo]
Aliquot 25µl Glycerol (pre-heated to 65ºC) to a new tube and then equilibrated to RT. 25µl of annealed oligo is mixed with glycerol. The annealed adapters can be immediately used for and stored at -20ºC.
?. [Fixation]
Spin down cells, wash cells with 0.5ml PBS-2RI, count alive cells with Trypan blue, resuspend cells with cold PBS-2RI at concentration of 1 million cells/ml.  AB1PBS-2RIVolume (µl)21× PBS200037.5% BSA10.74Enzymatic RI55SUPERase RI2.5
AB1PBS-2RIVolume (µl)21× PBS200037.5% BSA10.74Enzymatic RI55SUPERase RI2.5
?. [Fixation]
For each ml of cells in PBS-2RI, add 66.7µl of 1.6% or 3.2% FA (final concentration 0.1-0.2% FA, 0.1% for cell line, 0.2% for tissue) at RT, mix. Keep at RT for 5 min.
?. [Fixation]
For each ml of cells, quench fixation by adding 56.1µl of 2.5M Glycine, 50µl of 1M Tris pH 8.0, 13.3µl of 7.5% BSA, pipette mix. Keep on ice for 5 min.
?. [Fixation]
Spin down, remove supernatant, add 0.5ml PBS-2RI without disturbing the cell pellet.
?. [Fixation]
Spin down, remove supernatant, add 0.5ml PBS-2RI without disturbing the cell pellet.
?. [Fixation]
Resuspend cells to 50µl PBS-2RI.  AB1NIB-RIVolume (µl)2NIB80003Enzymatic RI204SUPERase RI2057.5% BSA42.7
AB1NIB-RIVolume (µl)2NIB80003Enzymatic RI204SUPERase RI2057.5% BSA42.7
If starting with nuclei, replace PBS-2RI in this section with NIB-RI
?. [ATAC]
Assemble transposome by mixing Tn5, dilution buffer and annealed transposition adapter oligo. Incubate at RT for 30min. The assembled transposome could be stored at -20ºC for up to 2 weeks.
?. [ATAC]
Dilute cells with PBS-RI to 2 million/ml, aliquot 5×N µl of diluted sample to a new tube. For example, for 100k cells, N=100k/10k =10, aliquot 5×10µl=50µl cells to a new tube.
?. [ATAC]
Add 42.5×N of 1𝗑 TB to sample, incubate at RT for 10min.
?. [ATAC]
Add 2.5×N µl of assembled Tn5 to sample, mix well by pipetting.  AB1TransposomeVolume (µl) 21× home-made Tn50.625×N3Dilution Buffer0.625×N4annealed transposition adapter with glycerol1.25×N5Total2.5×N
AB1TransposomeVolume (µl) 21× home-made Tn50.625×N3Dilution Buffer0.625×N4annealed transposition adapter with glycerol1.25×N5Total2.5×N
?. [ATAC]
Aliquot sample into 50µl per well in 96 well-plate, seal plate, shake at 500rpm for 30min, 37C
?. [ATAC]
Pool sample, spin down, add 0.5ml NIB-RI, spin down
?. [ATAC]
Resuspend cells in 60µl NIB-RI
?. [ATAC]
Calculate the number of required ATAC reactions. No. samples (N)=no. cells/10,000. For example, for 100k cells, N=100k/10k =10.  Prepare 2× TB and 1× TB accordingly. Critical: The 2× TB should be prepared freshly.  AB12× TB bufferVolume (µl)20.2M Tris-acetate33035M K-acetate26.441M Mg-acetate20510% NP-40206100% DMF3207H2O283.68Total1000  AB11× TBNo. samples (N)22× TB25×N3H2O16.45×N4PIC0.2×N5Enzymatic In0.85×N6Total42.5×N  AB1PBS-RIVolume (µl)21× PBS4003Enzymatic RI1
AB12× TB bufferVolume (µl)20.2M Tris-acetate33035M K-acetate26.441M Mg-acetate20510% NP-40206100% DMF3207H2O283.68Total1000
AB11× TBNo. samples (N)22× TB25×N3H2O16.45×N4PIC0.2×N5Enzymatic In0.85×N6Total42.5×N
AB1PBS-RIVolume (µl)21× PBS4003Enzymatic RI1
?. [RT]
Add 240µl RT mix to 60µl cells/NIB, aliquot 50µl to 6 PCR wells. Run RT thermal cycles. At the same time, start thawing the oligo plate at RT.  AB1Reverse transcription (RT) mixVolume (µl)25× RT buffer703Enzymatics RNase Inhibitor2.194SUPERase RI4.385dNTPs17.56100µM RT Primer357H2O10.94850% PEG1059Maxima H Minus Reverse Transcriptase (add right before RT reaction)3510Total280  ABC150ºC10min 28ºC12 s3 cycles315ºC45 s420ºC45 s530ºC30 s642ºC2 min750ºC3 min850ºC5 min 9Total37 min
AB1Reverse transcription (RT) mixVolume (µl)25× RT buffer703Enzymatics RNase Inhibitor2.194SUPERase RI4.385dNTPs17.56100µM RT Primer357H2O10.94850% PEG1059Maxima H Minus Reverse Transcriptase (add right before RT reaction)3510Total280
ABC150ºC10min 28ºC12 s3 cycles315ºC45 s420ºC45 s530ºC30 s642ºC2 min750ºC3 min850ºC5 min 9Total37 min
?. [RT]
Pool samples, spin down.
It is normal that the pellet sometimes looks bigger than before.
?. [RT]
Wash with 500µl NIB, spin down, and resuspend with 1152µl NIB-RI.
?. [Hybridization-ligation]
Thaw oligo plates to RT before hybridization.
?. [Hybridization-ligation]
Pipette mix 1152µl sample with 3456µl hybridization buffer, keep sample at RT, aliquot 40µl of mixture to Round 1 plate, pipette mix, and shake at 300rpm for 30min, RT  ABCD1Hybridization bufferStock conc.Final conc.Volume (µl)2WaterN/AN/A2761.93T4 ligase buffer 10×1×5764SUPERase RI20U/µl0.05U/µl14.45Enzymatics RI40U/µl0.32U/µl46.08610% NP4010%0.1%57.607Total  3456
ABCD1Hybridization bufferStock conc.Final conc.Volume (µl)2WaterN/AN/A2761.93T4 ligase buffer 10×1×5764SUPERase RI20U/µl0.05U/µl14.45Enzymatics RI40U/µl0.32U/µl46.08610% NP4010%0.1%57.607Total  3456
?. [Hybridization-ligation]
Add 10µl Blocking oligo 1 to each well, changing tips between rows. Set a multichannel to 40 µL and pipette each well up and down 4-5 times., and shake at 300rpm for 30min, RT  ABC1Blocking oligo 1Stock conc.Volume (µl)2Round_1_blocking100µM2533T4 ligase buffer10×2114Water 6875Total 1152
ABC1Blocking oligo 1Stock conc.Volume (µl)2Round_1_blocking100µM2533T4 ligase buffer10×2114Water 6875Total 1152
?. [Hybridization-ligation]
Pool samples, pipette mix in the reservoir. Aliquot 50µl of mixture to Round 2 plate, pipette mix, and shake at 300rpm for 30min, RT
?. [Hybridization-ligation]
Add 10µl Blocking oligo 2 to each well, changing tips between rows. Set a multichannel to 40 µL and pipette each well up and down 4-5 times., and shake at 300rpm for 30min, RT.  ABC1Blocking oligo 2Stock conc.Volume (µl)2Round_2_blocking100µM3043T4 ligase buffer10×2114Water 6375Total 1152
ABC1Blocking oligo 2Stock conc.Volume (µl)2Round_2_blocking100µM3043T4 ligase buffer10×2114Water 6375Total 1152
?. [Hybridization-ligation]
Pool samples, pipette mix in the reservoir. Aliquot 60µl of mixture to Round 3 plate, pipette mix, shake at 300rpm for 30min, RT.
?. [Hybridization-ligation]
Add 10µl Blocking oligo 3 to each well, changing tips between rows. Set a multichannel to 40 µL and pipette each well up and down 4-5 times., and shake at 300rpm for 30min, RT.  ABC1Blocking oligo 3Stock conc.Volume (µl)2Round_3_blocking100µM2653Triton X-1005%234Water 8645Total 1152
ABC1Blocking oligo 3Stock conc.Volume (µl)2Round_3_blocking100µM2653Triton X-1005%234Water 8645Total 1152
?. [Hybridization-ligation]
Pool samples, spin down, remove supernatant, wash with 1ml NIB-RI.
?. [Hybridization-ligation]
Spin down, remove supernatant, resuspend and wash with 1ml NIB-RI.
?. [Hybridization-ligation]
Spin down, remove supernatant, resuspend to 80µl NIB-RI.
?. [Hybridization-ligation]
Mix sample with 320µl Ligation mix, aliquot 50µl to 8 PCR tubes, shake at 300rpm for 30min, RT.  AB1Ligation mixVolume (µl)2Enzymatics RI3.23SUPERase RI1410× ligation buffer405T4 Ligase 400U/µl206H2O251.8710% NP4048Total320
AB1Ligation mixVolume (µl)2Enzymatics RI3.23SUPERase RI1410× ligation buffer405T4 Ligase 400U/µl206H2O251.8710% NP4048Total320
?. [Hybridization-ligation]
Gently resuspend with 1 mL NIB-RI and filter through 40 µm strainer (Flowmi) into a new 1.5 mL tube.
?. [Hybridization-ligation]
Spin, discard supernatant and resuspend to 50µl in NIB-RI.
?. [Hybridization-ligation]
Count cells with Trypan blue. Should now be nuclei that will stain with Trypan. Expect ~10-20K if started from 100K cells. Decide number of cells to be sequenced in each sub-library (generally 1-2K for troubleshooting/QC or 10K-20K for a full run).
optional pause point, may store product at -80ºC for at least a few days. We haven’t extensively tested this. Freezing extra nuclei at this stage at -80°C will usually result in good RNA quality but reduced ATAC yield.
?. [Reverse crosslinking-pull down]
Assuming N of 25µl sub-libraries are prepared. For each tube add 25µl 2× RCB, 2µl proteinase K (20mg/ml stock), 1µl SUPERase RI, 55ºC, 1h on thermal cycler.
optional stopping point, may store product at -80ºC for at least a few days. We haven’t extensively tested this. Freezing extra nuclei at this stage at -80°C will usually result in good RNA quality but reduced ATAC yield.
?. [Reverse crosslinking-pull down]
Mix with 2.5µl of 100mM PMSF (dissolved in IPA) and incubate at RT for 10min. At the same time, prepare following buffers.
?. [Reverse crosslinking-pull down]
Remove supernatant, wash twice with 100×N µl B&W-T without RI.
?. [Reverse crosslinking-pull down]
Remove supernatant, wash once with 100×N µl B&W-T/RI, and resuspend beads in 50×N µl 2× B&W/RI.  AB11× B&W-T/RIVolume/µl21× B&W-T400×(N+1)3SUPERase RI4×(N+1)  AB12× B&W/RIVolume/µl22× BW100×(N+1)3SUPERase RI2×(N+1)
AB11× B&W-T/RIVolume/µl21× B&W-T400×(N+1)3SUPERase RI4×(N+1)
AB12× B&W/RIVolume/µl22× BW100×(N+1)3SUPERase RI2×(N+1)
?. [Reverse crosslinking-pull down]
Add 50µl of beads to each sample, rotate at 10rpm, RT, 60min.
?. [Reverse crosslinking-pull down]
Put on a magnetic rack, transfer supernatant containing chromatin fragments to a new tube for library preparation later! The supernatant is stable at RT for a few hours.
?. [Reverse crosslinking-pull down]
Wash cDNA/RNA-bound beads with 100µl 1× B&W-T/RI, RT, three times.
?. [Reverse crosslinking-pull down]
Put the sample on the magnetic rack, wash with 100µl 1× STE/RI, without resuspending beads.  AB11× STE/RIVolume/µl2STE200×(N+1)3SUPERase RIN+1
AB11× STE/RIVolume/µl2STE200×(N+1)3SUPERase RIN+1
?. [ATAC library preparation]
ATAC library and RNA library are usually performed in parallel.
?. [ATAC library preparation]
Clean sample by Qiagen Minelute column with 500µl DNA binding buffer (Zymo) or PB buffer (Qiagen), elute to 11µl EB, elute again with additional 11µl EB (22µl in total).
Zymo DNA clean up column may result in inconsistent results between samples.
?. [ATAC library preparation]
Mix ~20µl sample with 29µl PCR mix, 1µl of 25µM Ad1.xx primer and H2O, run PCR for 5 cycles.  AB1ATAC PCR mixVolume (µl)2NEBNext 2× mix25×N3P7 primer 25µMN4H2O3×N5Total29×N  ABC1ATAC PCR 272ºC5 min 1 cycle398ºC30 s1 cycle498ºC10 s5 cycles565ºC30 s672ºC60 s
AB1ATAC PCR mixVolume (µl)2NEBNext 2× mix25×N3P7 primer 25µMN4H2O3×N5Total29×N
ABC1ATAC PCR 272ºC5 min 1 cycle398ºC30 s1 cycle498ºC10 s5 cycles565ºC30 s672ºC60 s
?. [ATAC library preparation]
qPCR: 5µl sample + 10µl qPCR mix, calculate the number of cycles needed to reach ⅓ of the plateau fluorescence (0.33 Ct).  ABC1qPCR 298ºC30 s1 cycle398ºC10 s20 cycles465ºC30 s572ºC60 s  AB1ATAC qPCR mixVolume (µl)2NEBNext 2× mix5×N3Ad1.1 25µM0.2×N4P7 25µM0.2×N510× SYBRgreen0.9×N6H2O3.7×N7Total10×N
ABC1qPCR 298ºC30 s1 cycle398ºC10 s20 cycles465ºC30 s572ºC60 s
AB1ATAC qPCR mixVolume (µl)2NEBNext 2× mix5×N3Ad1.1 25µM0.2×N4P7 25µM0.2×N510× SYBRgreen0.9×N6H2O3.7×N7Total10×N
?. [ATAC library preparation]
check qPCR product size on gel, expect smear with average size ~400bp. Nucleosome pattern may or may not show up
10,000× SYBRgreen was diluted by water to 100×, and stored in black tube at -20ºC. 100× SYBR was further diluted by water to 10× freshly.
?. [ATAC library preparation]
Run additional PCR cycles for the rest of sample (~45µl), N=0.33 Ct cycles.  ABC1Additional PCR 298ºC30s398ºC10s0.33 Ct cycles465ºC30s572ºC60s
ABC1Additional PCR 298ºC30s398ºC10s0.33 Ct cycles465ºC30s572ºC60s
?. [ATAC library preparation]
Expect ~11-13 additional cycles for 2K cells or ~8-10 for 10K cells.
?. [ATAC library preparation]
Clean sample by Zymo DNA clean up kit (225µl binding buffer or 250µl if skipped qPCR), elute to 6µl EB, elute again with additional 6µl EB (~10µl in total). Store the libraries at -20°C until quantification.
?. [RNA library preparation (Template switching)]
Remove all supernatant, resuspend beads in 50µl Template switch mix, be careful to avoid beads to dry.
?. [RNA library preparation (Template switching)]
Rotate samples at RT for 30min at 10rpm.
?. [RNA library preparation (Template switching)]
Incubate at 42ºC for 90min, shaking at 300rpm, resuspend beads every 30min by pipetting mix up and down.  ABC1Template switch mixVolume (µl)Final conc.2H2O1.25×(N+1) 350% PEG 600015×(N+1)15%45× Maxima RT buffer10×(N+1)1𝗑5Ficoll PM-400 (20%)10×(N+1)4%610mM dNTPs, each5×(N+1)1mM7NxGen RNase Inhibitor5×(N+1)0.1𝗑8100µM TSO (BC_0127)1.25×(N+1)2.5µM9Maxima RT Rnase H Minus (add last)2.53×(N+1) 10Total50×(N+1)
ABC1Template switch mixVolume (µl)Final conc.2H2O1.25×(N+1) 350% PEG 600015×(N+1)15%45× Maxima RT buffer10×(N+1)1𝗑5Ficoll PM-400 (20%)10×(N+1)4%610mM dNTPs, each5×(N+1)1mM7NxGen RNase Inhibitor5×(N+1)0.1𝗑8100µM TSO (BC_0127)1.25×(N+1)2.5µM9Maxima RT Rnase H Minus (add last)2.53×(N+1) 10Total50×(N+1)
?. [cDNA amplification]
Mix each sample with 100µl H2O, magnetize and discard supernatant, wash with 200µl STE, without resuspend beads.
?. [cDNA amplification]
Mix 55µl PCR mix with beads, transfer sample to PCR plate.
?. [cDNA amplification]
Run PCR for 5 cycles, with sample against magnetic rack, transfer supernatant to new PCR wells.  AB1cDNA PCR mixVolume (µl)2Kapa Hifi 2× mix27.5×(N+1)3RNA PCR primer 25µM0.88×(N+1)4P7 primer 25µM0.88×(N+1)5H2O23.24×(N+1)6Total55×(N+1)  AB1PCR 295ºC3min398ºC20s465ºC45s572ºC3min
AB1cDNA PCR mixVolume (µl)2Kapa Hifi 2× mix27.5×(N+1)3RNA PCR primer 25µM0.88×(N+1)4P7 primer 25µM0.88×(N+1)5H2O23.24×(N+1)6Total55×(N+1)
AB1PCR 295ºC3min398ºC20s465ºC45s572ºC3min
?. [cDNA amplification]
qPCR: For each sublibrary, mix 2.5µl sample with 7.5µl qPCR mix, run qPCR protocol and calculate 0.33 Ct.  AB1RNA qPCR mixVolume (µl)2Kapa Hifi 2× mix3.75×(N+1)3RNA PCR primer 25µM0.12×(N+1)4P7 25µM0.12×(N+1)5EVAgreen 20×0.5×(N+1)6H2O3.01×(N+1)7Total7.5×(N+1)  ABC1qPCR 295ºC3min398ºC20s20 cycles465ºC20s572ºC3min
AB1RNA qPCR mixVolume (µl)2Kapa Hifi 2× mix3.75×(N+1)3RNA PCR primer 25µM0.12×(N+1)4P7 25µM0.12×(N+1)5EVAgreen 20×0.5×(N+1)6H2O3.01×(N+1)7Total7.5×(N+1)
ABC1qPCR 295ºC3min398ºC20s20 cycles465ºC20s572ºC3min
?. [cDNA amplification]
check qPCR product size on gel, expect smear with average size > 1kb.
?. [cDNA amplification]
Run additional PCR cycles for the rest of the sample (~50µl), N=0.33 Ct cycles.  ABC1Additional cycles 295ºC3min398ºC20s0.33 Ct cycles465ºC20s572ºC3min
ABC1Additional cycles 295ºC3min398ºC20s0.33 Ct cycles465ºC20s572ºC3min
For cell line, expect 6-7 additional cycles for 2k cells, ~4 additional cycles for 10k cells. More cycles are expected for primary cells or nuclei.
?. [cDNA amplification]
Purify each sample with 42µl AMpure beads (0.8×) for cell line sample or 31.5µl AMpure beads (0.6×) for primary samples, elute cDNA to 10µl EB. Store the libraries at -20°C.
?. [Tagmentation]
Quantify cDNA concentration by Qubit using 1µl cDNA. Ideally, dilute 50ng cDNA to 5ng/µl in H2O.
Use nanodrop may result in inaccurate quantificationExpect > 50ng cDNA. If cDNA amount is low, it can get away with tagmenting 20ng cDNA; in this case, adjust the volume of H2O and cDNA accordingly.
?. [Tagmentation]
Assemble in-house produced Tn5, prepare transposome by mixing Tn5, dilution buffer and annealed oligo, and incubate the mix at RT for 30min.  AB1Assemble transposomeVolume (µl) 21× Tn51.25×(N+1)3Dilution Buffer1.25×(N+1)4annealed tagmentation adapter with glycerol2.5×(N+1)5Total5×(N+1)
AB1Assemble transposomeVolume (µl) 21× Tn51.25×(N+1)3Dilution Buffer1.25×(N+1)4annealed tagmentation adapter with glycerol2.5×(N+1)5Total5×(N+1)
?. [Tagmentation]
Mix 10µl of 5ng/µl cDNA with 10µl H2O, 25µl TD buffer (Illumina) and 5µl assembled Tn5. Incubate at 55ºC for 5min on a thermal cycler.
?. [Tagmentation]
Purify tagmented library with QIagen minelute column using 250µl PB buffer, elute twice with 11µl EB each time (22µl in total).
?. [Tagmentation]
Add 29µl of post-tagmentation PCR mix with 1µl tube specific Ad 1.xx primer, amp 7 cycles.  AB1Post-tagmentation PCR mixVolume (µl)2Sample in EB20 each3NEBNext 2× mix25×(N+1)425µM P71×(N+1)525µM Ad 1.xx1×(N+1)6H2O3×(N+1)7Total50×(N+1)  ABC1Tagmentation PCR 272ºC5min398ºC30s498ºC10s7 cycles565ºC30s672ºC60s
AB1Post-tagmentation PCR mixVolume (µl)2Sample in EB20 each3NEBNext 2× mix25×(N+1)425µM P71×(N+1)525µM Ad 1.xx1×(N+1)6H2O3×(N+1)7Total50×(N+1)
ABC1Tagmentation PCR 272ºC5min398ºC30s498ºC10s7 cycles565ºC30s672ºC60s
?. [Tagmentation]
Purify each sample with 35µl AMpure beads (0.7×), elute library to 10µl EB. Store the libraries at -20°C until quantification.
?. [Library quantification and sequencing]
Recommend quantifying with KAPA qPCR kit and pool to 4nM. Expect > 30nM, average library size ~350bp with minimal adapter dimer.
?. [Library quantification and sequencing]
We typically sequence libraries with a Next-seq High output 150 cycle kit. The sequencing read configuration is 50×99×8×10 for the RNA-seq library; 30×99×8×30 for the pooled ATAC/RNA or ATAC-seq library. In general, ~25k reads per cell per library type for ~50% duplication rate and ~200k reads per cell per library type for ~90% duplication rate
?. [Buffer preparation]
Prepare following buffer and store at 4°C  ABC1Nuclei Isolation Buffer (NIB)Volume (ml)Final conc.21M Tris HCl pH 7.50.510mM35M NaCl0.110mM41M MgCl20.153mM510% NP400.50.1%6H2O48.75 7Total50   ABC12× BWVolume (ml)2× conc.21M Tris 8.00.510mM35M NaCl202M40.5M EDTA0.11mM5H2O29.4 6Total50   ABC11𝗑 B&W-TVolume (ml)1𝗑 conc.21M Tris 8.00.255mM35M NaCl101M40.5M EDTA0.050.5mM5100% Tween 200.0250.05%6H2O39.675 7Total50   ABC1Oligo resuspension buffer (IDTE)Volume (ml)1𝗑 conc.21M Tris 8.00.510mM30.5M EDTA0.010.1mM4H2O49.5 5Total50   ABC1Oligo annealing buffer (STE)Volume (ml)1𝗑 conc.21M Tris 8.00.510mM35M NaCl0.550mM40.5M EDTA0.11mM5H2O48.9 6Total50   ABC1Dilution BufferVolume (µl)Final conc.2100% glycerol (use wide bore tips)50050%31M Tris pH 7.55050mM45M NaCl20100mM55mM EDTA200.1mM6100mM DTT101mM710% NP-40100.1%8H2O390 9Total1000
ABC1Nuclei Isolation Buffer (NIB)Volume (ml)Final conc.21M Tris HCl pH 7.50.510mM35M NaCl0.110mM41M MgCl20.153mM510% NP400.50.1%6H2O48.75 7Total50
ABC12× BWVolume (ml)2× conc.21M Tris 8.00.510mM35M NaCl202M40.5M EDTA0.11mM5H2O29.4 6Total50
ABC11𝗑 B&W-TVolume (ml)1𝗑 conc.21M Tris 8.00.255mM35M NaCl101M40.5M EDTA0.050.5mM5100% Tween 200.0250.05%6H2O39.675 7Total50
ABC1Oligo resuspension buffer (IDTE)Volume (ml)1𝗑 conc.21M Tris 8.00.510mM30.5M EDTA0.010.1mM4H2O49.5 5Total50
ABC1Oligo annealing buffer (STE)Volume (ml)1𝗑 conc.21M Tris 8.00.510mM35M NaCl0.550mM40.5M EDTA0.11mM5H2O48.9 6Total50
ABC1Dilution BufferVolume (µl)Final conc.2100% glycerol (use wide bore tips)50050%31M Tris pH 7.55050mM45M NaCl20100mM55mM EDTA200.1mM6100mM DTT101mM710% NP-40100.1%8H2O390 9Total1000
?. [Buffer preparation]
Prepare following buffer and store at room temperature.  ABC12× RCBVolume (ml)2× conc.21M Tris 8.01100mM35M NaCl0.2100mM420% SDS0.20.40%5H2O8.58 6Total10 PEG 6000 50%: mix equal mass of PEG6000 and H2O, dissolve PEG by heating to 65ºC for 4min and cooling down to RT.
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
ABC12× RCBVolume (ml)2× conc.21M Tris 8.01100mM35M NaCl0.2100mM420% SDS0.20.40%5H2O8.58 6Total10
?. [RT]
Optional pause point, after moving supernatnt, store the nuclei pellet at -80°C