title: GSM4163452: 190305_mCTseq_hs_21yr_BA10_UMB5577_1_UMB5577_2_A1_AD007_indexed; Homo sapiens; OTHER
library_construction_protocol: Nuclei isolation from cultured cells for snmCT-seq: Cell pellets containing 1 million cells were resuspended in 600 µl NIBT [250 mM Sucrose, 10 mM Tris-Cl pH=8, 25 mM KCl, 5mM MgCl2, 0.1% Triton X-100, 1mM DTT, 1:100 Proteinase inhibitor (Sigma-Aldrich P8340), 1:1000 SUPERaseIn RNase Inhibitor (ThermoFisher Scientific AM2694), 1:1000 RNaseOUT RNase Inhibitor (ThermoFisher Scientific 10777019)]. The lysate was transferred to a pre-chilled 2 ml dounce homogenizer (Sigma-Aldrich D8938) and dounced using loose and tight pestles for 20 times each. The lysate was then mixed with 400 µl of 50% Iodixanol (Sigma-Aldrich D1556) and gently pipetted on top of 500 µl 25% Iodixanol cushion. Nuclei were pelleted by centrifugation at 10,000 x g at 4°C for 20 min using a swing rotor. The pellet was resuspended in 2 ml of DPBS supplemented with 1:1000 SUPERaseIn RNase Inhibitor and 1:1000 RNaseOUT RNase Inhibitor. Hoechst 33342 was added to the sample to a final concentration of 1.25 nM and incubated on ice for 5 min for nuclei staining. Nuclei were pelleted by 1,000 x g at 4°C for 10 min and resuspended in 1 ml of DPBS supplemented with RNase inhibitors.   Nuclei isolation from human brain tissues and GpC methyltransferase treatment  Brain tissue samples were ground in liquid nitrogen with cold mortar and pestle, and then aliquoted and store at -80°C. Approximately 100mg of ground tissue was resuspended in 3 ml NIBT (250 mM Sucrose, 10 mM Tris-Cl pH=8, 25 mM KCl, 5mM MgCl2, 0.2% IGEPAL CA-630, 1mM DTT, 1:100 Proteinase inhibitor (Sigma-Aldrich P8340), 1:1000 SUPERaseIn RNase Inhibitor (ThermoFisher Scientific AM2694), 1:1000 RNaseOUT RNase Inhibitor (ThermoFisher Scientific 10777019)). The lysate was transferred to a pre-chilled 7 ml dounce homogenizer (Sigma-Aldrich D9063) and dounced using loose and tight pestles for 40 times each. The lysate was then mixed with 2 ml of 50% Iodixanol (Sigma-Aldrich D1556) to generate a nuclei suspension with 20% Iodixanol. Gently pipet 1 ml of the nuclei suspension on top of 500 µl 25% Iodixanol cushion in each of the 5 freshly prepared 2ml microcentrifuge tubes. Nuclei were pelleted by centrifugation at 10,000 x g at 4°C for 20 min using a swing rotor. The pellet was resuspended in 1ml of DPBS supplemented with 1:1000 SUPERaseIn RNase Inhibitor and 1:1000 RNaseOUT RNase Inhibitor. A 10 µl aliquot of the suspension was taken for nuclei counting using a Biorad TC20 Automated Cell Counter. One million nuclei aliquots were pelleted by 1,000 x g at 4°C for 10 min and resuspended in 200 µl of GpC methyltransferase M.CviPI (NEB M0227L) reaction containing 1X GC Reaction Buffer, 0.32 nM S-Adenoslylmethionime, 80U 4U/µl M.CviPI, 1:100 SUPERaseIn RNase Inhibitor and 1:100 RNaseOUT RNase Inhibitor and incubated at 37°C for 8 min. The reaction was stopped by adding 800 µl of ice-cold DPBS with 1:1000 RNase inhibitors and mixing. Hoechst 33342 was added to the sample to a final concentration of 1.25 nM and incubated on ice for 5 min for nuclei staining. Nuclei were pelleted by 1,000 x g at 4°C for 10 min, resuspended in 900 µl of DPBS supplemented with 1:1000 RNase inhibitors and 100 µl of 50mg/ml UltrapureTM BSA (Ambion AM2618) and incubated on ice for 5 min for blocking. Neuronal nuclei were labeled by adding 1 µl of AlexaFluor488-conjugated anti-NeuN antibody (clone A60, MilliporeSigma MAB377XMI) for 20 min. For snATAC-seq, nuclei were isolated from frozen mouse brain sections and permeabilized. Nuclei were tagmented with Tn5 in 96 wells to introduce a first barcode (T5 and T7). After pooling, 20 nuclei were sorted into 768 wells and a second barcode (i5 and i7) was introduced by PCR. Reverse transcription for snmC2T-seq Single cells or single nuclei were sorted into 384-well PCR plates (ThermoFisher 4483285) containing 1 µl mCT-seq reverse transcription reaction per well. The mCT-seq reverse transcription reaction contained 1X Superscript II First-Strand Buffer, 5mM DTT, 0.1% Triton X-100, 2.5 mM MgCl2, 500 µM each of 5'-methyl-dCTP (NEB N0356S), dATP, dTTP and dGTP, 1.2 µM dT30VN_4 oligo-dT primer (5'-AAGCAGUGGUAUCAACGCAGAGUACUTTTTTUTTTTTUTTTTTUTTTTTUTTTTTVN-3' was used the cultured cell experiments; 5'-/5SpC3/AAGCAGUGGUAUCAACGCAGAGUACUTTTTTUTTTTTUTTTTTUTTTTTUTTTTTVN-3' was used for human brain experiments), 2.4 µM TSO_3 template switching oligo (/5SpC3/AAGCAGUGGUAUCAACGCAGAGUGAAUrGrG+G), 1U RNaseOUT RNase inhibitor, 0.5 U SUPERaseIn RNase inhibitor, 10U Superscript II Reverse Transcriptase (ThermoFisher 18064-071). For snmC2T-seq, the reaction further included 2 µM N6_2 random primer (/5SpC3/AAGCAGUGGUAUCAACGCAGAGUACNNNNNN). After sorting, the PCR plates were vortexed and centrifuged at 2000 x g. The plates were placed in a thermocycler and incubated using the following program: 25°C for 5 min, 42°C for 90min, 70°C 15min followed by 4°C. cDNA amplification for snmC2T-seq 3 µl of mCT-seq cDNA amplification mix was added into each mCT-seq reverse transcription reaction. mCT-seq cDNA amplification reaction contains 1X KAPA 2G Buffer A, 600 nM ISPCR23_2 PCR primer (/5SpC3/AAGCAGUGGUAUCAACGCAGAGU), 0.08U KAPA2G Robust HotStart DNA Polymerase (5 U/μL, Roche KK5517). PCR reactions were performed using a thermocycler with the following conditions: 95°C 3min -> [95°C 15 sec -> 60°C 30 sec -> 72°C 2min] -> 72°C 5min -> 4°C. The cycling steps were repeated for 12 cycles for scmCT-seq using H1 or HEK293 cells, 15 cycles for snmCT-seq using H1 or HEK293 cells and 14 cycles for snmC2T-seq using human brain tissues. Digestion of unincorporated DNA oligos for snmCT-seq For scmCT-seq and snmCT-seq using H1 and HEK293 cells, 1 µl uracil cleavage mix was added to into cDNA amplification reaction. Each 1 µl uracil cleavage mix contains 0.25 µl Uracil DNA Glycosylase (Enzymatics G5010) and 0.25 µl Endonuclease VIII (Enzymatics Y9080) and 0.5 µl Elution Buffer (Qiagen 19086). Unincorporated DNA oligos were digested at 37°C for 30 min using a thermocycler. We have found that Endonuclease VIII is dispensable for the digestion of unincorporated DNA oligos since the alkaline condition during the desulfonation step of bisulfite conversion can effective cleave abasic sites created by Uracil DNA Glycosylase (Greenberg, 2014). Therefore for snmC2T-seq using human brain tissues, each cDNA amplification reaction was treated with 1µl uracil cleavage mix containing 0.5 µl Uracil DNA Glycosylase (Enzymatics G5010-1140) and 0.5 µl Elution Buffer (Qiagen 19086). For snATAC-seq, nuclei were tagmented with Tn5 in 96 wells to introduce a first barcode (T5 and T7). After pooling, 20 nuclei were sorted into 768 wells and a second barcode (i5 and i7) was introduced by PCR. Libraries were sequenced on a HiSeq2500 (Illumina) with following read lengths: 50 + 14 + 14 + 50 (Read1 + Index1 + Index2 + Read2).
attributes.tag: GEO Accession
attributes.value: GSM4163452