title: GSM7876401: M1_donor3_rep1_Multiome H3K27ac; Homo sapiens; OTHER
library_name: GSM7876401
library_construction_protocol: nuclei preparation protocol: Cells from frozen tissue were extracted by douncing in douncing buffer (0.25 M sucrose (Sigma, S7903), 25 mM KCl (Sigma, P9333), 5 mM MgCl2, 10 mM Tris-HCl pH 7.4, 1 mM DTT (Sigma, D9779), 1× Protease Inhibitor, 0.5 U µl−1 RNase OUT, 0.5 U µl−1 SUPERase Inhibitor). The cell suspension was filtered through a 30-µm Cell-Tric (Sysmex) to remove debris and spun down for 10min at 300g, 4 °C. Cell pellets were washed once with douncing buffer, spun down again, and resuspended in cold nuclei permeabilization buffer for 10mins. Permeablized nuclei were pelleted by centrifuge for 10 min at 1,000g, 4 °C, and washed with sort buffer (1× PBS (Gibco, 10010023), 1×Protease Inhibitor (Roche, 05056489001), 0.5 U µl−1 RNase OUT (Invitrogen, 10777-019), 0.5 U µl−1 SUPERase Inhibitor (Invitrogen, AM2694), 1mM EDTA (Invitrogen, 15575020), 1% BSA (Sigma, A1595)). Nuclei were resuspended in sort buffer and stained with  μM 7-AAD (Invitrogen, A1310) for 10mins on ice, and proceed to Fluorescence-activated cell sorting (FACS) with an SH800 cell sorter (Sony) to isolate of single nucleus. Nuclei were collected in collection buffer (1× PBS (Gibco, 10010023), 5×Protease Inhibitor (Roche, 05056489001), 2.5 U µl−1 RNase OUT (Invitrogen, 10777-019), 2.5 U µl−1 SUPERase Inhibitor (Invitrogen, AM2694), 1mM EDTA (Invitrogen, 15575020), 5% BSA (Sigma, A1595)) at 5 °C. After centrifugation for 10min at 750g at 4 °C. Nuclei were washed twice with sort buffer and counted on an RWD C100-pro fluorescence cell counter with DAPI staining. For each histone modification, around 0.5 million sorted nuclei were aliquoted and proceeded to Paired-Tag experiments. DNA and RNA libraries were generated according to the Chromium Single Cell ATAC Library kit manual with increased number of amplification cycles (12-13 cycles) for DNA library. All sequencing was performed with an Illumina Nextseq2000 sequencer.