GSM8594443: reaction_6_RNA; Mus musculus; RNA-Seq
Nucleus pellets were suspended in 110 µL nuclei buffer (PBS; w/o MgCl₂ and CaCl₂ [20012068; Invitrogen]), 1 % BSA (SRE0036-25 ml; Sigma), 2 mM MgCl2 (M1028-100 ml; Sigma), and 0.1 % Protector RNase inhibitor 40 U/μl (3335399001; Sigma), and the solution was passed through a 40 μm strainer (43-10040-40; pluriSelect life Science, Germany). For each sample, 10 µL was recovered and mixed with 10 µL trypan blue solution. 93595-50 ml, Sigma) for counting nuclei in a counting chamber. The remaining 100 µL were added 1 µL of individual TotalSeq-A antibodies (BioLegend, San Diego, CA), for a final concentration of 5 ng/µL to enable multiplexing. Samples were washed, centrifuged, re-suspended and stored. Using a SH800S Cell Sorter (SONY), 6,000 nuclei from each of the 24 samples was sorted. From two of the liver samples, only 3,005 and 4,015 nuclei could be recovered. Nuclei from four samples were mixed resulting in a total of six pools. Each pool containing 24,000 nuclei were loaded on a 10X reaction using 10X Genomics Chromium single-cell 3′ reagent kits (version 3.1). Single-cell cDNA libraries were produced according to the manufacturer's instructions and sequenced on an Illumina NovaSeq 6000 (Illumina, San Diego, CA) using 2x S1 flow cells