GSM6808301: s91C; Homo sapiens; RNA-Seq
A petri dish, 50 ml centrifuge tube, scalpel and forceps were precooled on dry ice before tissue samples were removed from -80°C and placed in the petri dish. Typical sample sizes ranged from 100-500 mg.  Tissue was cut into thin slices and transferred to centrifuge tubes. If processing multiple samples, cut tissue could be stored at -80°C until use.  Sample tubes were transferred to wet ice, 4 ml of ice-cold CST buffer (146 mM NaCl, 10 mM Tris-HCl pH 7.5, 1 mM CaCl, 21 mM MgCl2, 0.5% CHAPS (w/v), 0.01% BSA (w/v), 4 ul/ml SUPERaseIN, 4 ul/ml RNasein Plus, 1 cOmplete protease inhibitor tablet/10 ml) added and tubes placed on a rotator for 10 minutes at 4°C.  Samples were passed through 30 µm cell strainers (MACS SmartStrainer) into prechilled 15 ml collection tubes on ice. Sample tubes were rinsed with 2 ml ice cold PBS + 1% BSA, which was added to the cell strainer. Cell strainers were rinsed with an additional 2 ml ice cold PBS + 1% BSA and collection tubes centrifuged at 500g for 5 minutes at 4°C.  Supernatant was removed and pellet washed by resuspending in 10 ml ice cold PBS + 1% BSA, centrifugation at 500g for 5 minutes at 4°C, removal of supernatant and resuspension in 500 µl ice cold PBS + 1% BSA. A subsample of the nuclei preparation was incubated with DAPI (1 µg/ml) for 5 minutes, added to a haemocytometer and counted under a fluorescent microscope. Concentration of the nuclei was adjusted to ~1,000 cells/µl and used as input for analysis by 10X Chromium single cell gene expression. Chromium single cell gene expression (10X Genomics) was performed using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1, Chromium Next GEM Chip G Single Cell Kit and Single Index Kit T Set A according to the manufacturer's instructions starting with 20,000 nuclei as input. Resulting libraries were quantitated by TapeStation (Agilent), pooled at equimolar concentration and sequenced (Novogene (UK) Ltd or Genewiz GmbH) on an Illumina NovaSeq 6000 using a S4 Reagent Kit v1.5 to give ~30,000 reads/cell. 10X Genomics