GSM8724799: Test,Female,Rep2; Mus musculus; RNA-Seq
Fresh-frozen tissue samples were used for snRNAseq analysis. Tissue blocks embedded in optimal cutting temperature (OCT) compound (Tissue-Tek, Sankura; #4583) were sectioned into 20 µm thick curls (4–5 per sample) using a Leica CM1950 cryostat (Leica, Wetzlar, Germany). The sections were transferred into 5 mL tubes (Eppendorf, Hamburg, Germany), washed with ice-cold PBS (Thermo Fisher, #10010023), centrifuged at 400 × g for 2 minutes, and the supernatant was removed. The tissue was then resuspended in 1 mL ST buffer [146 mM NaCl, 10 mM Tris-HCl (pH 7.5), 1 mM CaCl2, and 21 mM MgCl2 in ultrapure water] containing 0.03% Tween-20 (Sigma Aldrich, #p-7949) and 0.1% BSA (New England Biolabs, B9000S), with or without 40 U/mL RNAse inhibitor (RNAse OUT, Thermo Fisher). The suspension was mechanically dissociated by pipetting 15 times with a 1 mL pipette, followed by a 5-minute incubation on ice. After the incubation, pipetting was repeated, and the reaction was stopped by adding 4 mL ST buffer (with or without RNAse inhibitor). The suspension was filtered twice through pre-wetted 70 µm mesh filters into a 50 mL conical tube, and the filter was washed with 5 mL ST buffer. The filtered sample was centrifuged at 500 × g for 5 minutes, and the pellet containing dissociated nuclei was resuspended in 100–400 µL ST buffer. The suspension was then passed through a 40 µm mesh filter, and nuclei were counted using a Neubauer chamber (Bulldog Bio, Portsmouth, NH) after staining with 50 µg/mL Hoechst 33342 (Thermo Fisher, H3570). A total of 0.9–1.5 × 10³ nuclei were loaded into ST buffer without RNAse inhibitor using a Chromium controller and Chromium reagents (10X Genomics). cDNA libraries were prepared following the manufacturer's instructions, with an additional cycle of cDNA amplification to account for the lower RNA content in nuclei compared to whole cells. The final sequencing libraries were quantified using the 2200 TapeStation system with D1000 and D5000 reagents (Agilent). The libraries were pooled and sequenced to achieve a target depth of >20,000 reads per cell for gene expression libraries, using NovaSeq 6000 (Illumina, San Diego, CA) with a minimum read length of 2 × 100 bp.