title: GSM7025963: FC_Droplet_Paired-Tag_H3K27me3_rep3_RNA; Mus musculus; OTHER
library_construction_protocol: Single-cell suspensions were prepared from frozen tissues 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 then filtered through a 30-µm Cell-Tric (Sysmex) for debris removal, and spun down for 10 min 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)) once. After resuspension in sort buffer, nuclei are stained with 2 μM 7-AAD (Invitrogen, A1310) for 10mins on ice, and proceed to Fluorescence-activated cell sorting (FACS) with an SH800 cell sorter (Sony) for isolation of single nucleus. Nuclei are 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, then immediately proceed to centrifuge for 10 min at 750g, 4 °C. Nuclei were washed twice with sort buffer and counted on an RWD C100-pro fluorescence cell counter with DAPI staining to better estimate the nuclei number. For each histone modification, around 0.5 million sorted nuclei were aliquoted and proceeded to Paired-Tag experiments. Final DNA and RNA libraries amplification were performed according to the Chromium Single Cell ATAC Library kit manual, except that we used an increased number of amplification cycles (in total 12-13) for DNA (histone modality) library.