GSM7520257: mESC_Mettl3KO_m6A-CT_scRNA-seq; Mus musculus; RNA-Seq
E14 mESCs were harvested, counted and centrifuged for 5 min at 350×g at room temperature. Aliquots of cells (~1,000,000 cells) were resuspended in 250 μL Nuclei Extraction Buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.020 % Triton X-100) and incubated on ice for 1 min. The mixture was diluted with 1 mL Nuclei Dilution Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor) and centrifuged for 5 min at 600×g at 4 °C. Nuclei were washed once with 1 mL Nuclei Dilution Buffer and counted. E14 EPSC and MEF nuclei were similarly isolated with the following conditions: 1 min on ice 0.015 % Triton X-100 for EPSC; 5 min on ice in 0.2 % NP-40 for MEF. Aliquots of nuclei (150,000 nuclei in total per assay) were incubated with 1 μg primary antibody (rabbit IgG or rabbit anti-m6A: MilliporeSigma, #ABE572) in 50 μL Antibody Binding Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) for 2 hours on rotator at 4 °C. Afterwards, nuclei were incubated with 1 μg secondary antibody (guinea pig anti-rabbit: Novus Biologicals, #NBP1-72763) in 50 uL Antibody Binding Buffer for 30 min on the nutator at room temperature. Nuclei were then centrifuged for 5 min at 600×g at room temperature and two rounds of washes with 200 μL Wash Buffer (20 mM HEPES pH7.5, 150 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) were performed. The nuclei were resuspended in 50 μL high-salt Binding Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) with 2.5 μL pAG-Tn5 (EpiCypher, #15-1117) and incubated for 1 hour on the nutator at room temperature. The nuclei were then washed twice with high-salt Wash Buffer (20 mM HEPES pH7.5, 300 mM NaCl, 1X Roche Complete Protease Inhibitor Cocktail, 0.1 U/μL RiboLock RNase inhibitor, 0.005 % Triton X-100) and resuspended in 50 μL Reverse Transcription & Tagmentation Buffer (1X SuperScript IV buffer along with, 1 M Betaine, 5 mM DTT, 2 μM Random Hexmer Primers, 0.5 mM each dNTP mix, 1X Roche Complete Protease Inhibitor Cocktail, 1 U/μL RiboLock RNase inhibitor, 4 U/μL SuperScript IV) for 2 hours at 37 °C. When reaction was done, the nuclei were resuspended with 50 μL cold Antibody Binding Buffer and placed on ice. All steps after nuclei extraction were performed in 0.2-ml tubes to minimize nuclei loss.  The nuclei were counted and diluted to a concentration based on the targeted nuclei number. Seven microliters of 10x Genomics ATAC Buffer B was added to 8 μl of nuclei, pipetting several times to mix. Most of the remaining steps were performed according to the Chromium Next GEM Single Cell Multiome protocol Rev E (10x Genomics), skipping the Step 1 'Transposition' and continuing from Step 2.0 'Generation & barcoding'. A qPCR cycle check was performed after pre-amplification step using 1:100 amplicon. Indexed CT libraries were prepared with the following PCR program: 45 sec at 98 °C, followed by 23 cycles (ESC IgG-CT) or 15 cycles (ESC and mixed cell-lines m6A-CT) of 15 sec at 98 °