GSM7433715: NCs, scRNAseq; Homo sapiens; RNA-Seq
For nuclei isolation, all following steps were performed on ice with pre-cooled buffers and centrifugation steps were performed at 4 oC. In brief, the snap-frozen samples were thawed using 500 µl ice-cold homogenization buffer [HB; consisting of 320 mM sucrose, 5 mM CaCl2, 3 mM magnesium acetate, 10 mM Tris pH 7.8, 0.1 mM EDTA, 0.1% NP40, 1X complete protease inhibitor (Roche), 1 mM β-mercaptoethanol and RNase-In inhibitor (Promega)] for 5 min to prevent the nuclei from rupturing. The samples were then homogenized with pestle A (5 gentle strokes) and pestle B (15 gentle strokes) and incubated on ice for 5 min.  The homogenates were filtered using FlowmiTM 70 µm cell strainers and the filtrates were centrifuged at 500g for 5 min. The resulting pellets were resuspended in 2.65 ml washing buffer (HB without NP40) and 2.65 ml gradient medium (5 mM CaCl2, 50% Optiprep, 3 mM magnesium acetate, 10 mM Tris pH 7.8, 1X complete protease inhibitor, 1 mM β-mercaptoethanol and RNase-In inhibitor) and the resulting solution was added gently on top of a cushion solution (29% Optiprep, 150 mM KCl, 30 mM MgCl2, 60 mM Tris pH 7.8 and  250 mM sucrose) for centrifugation at 7700 rpm for 30 min at 4 oC. The nuclei pellets were resuspended in 25 µl resuspension buffer (1X PBS, 2% BSA), filtered using FlowmiTM 40 µm cell strainers and performed nuclei counting using LUNA automated cell counter (Logos Biosystems) according to the manufacturer's instructions The diluted nuclei suspensions were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) (Next GEM Single Cell 3' library and Gel Bead Kit v3.1) according to the manufacturer's protocol (10x User Guide; CG000204, Revision D). Generation of gel beads in emulsion (GEMs), barcoding, GEM-RT cleanup, complementary DNA amplification and library construction were all performed according to the manufacturer's protocol. Individual sample quality was assessed using a Tapestation (Agilent). Qubit 2.0 (ThermoFisher Scientific) was used for library quantification before pooling libraries. The final library pool was sequenced on NextSeq2000 (Illumina) instrument using NextSeq 1000/2000 P3 kit v3 for 2 lanes of 100-base-pair paired-end reads.