GSM6285493: Tumor NF110; Homo sapiens; OTHER
Flash frozen archived tumor specimens were minced with sterile scalpel and mechanically dissociated with a dounce tissue grinder (size A and B, Sigma Aldrich) in ice cold lysis buffer (0.32 M sucrose, 5mM CaCl2, 3 mM MgAc2, 0.1 mM EDTA, 20 mM Tris-HCl, 40U/ml RNase inhibitor and 0.1% Triton X-100 in DEPC-treated water). After centrifuged at 800g for 10 mins and resuspended in 1m of wash buffer (1x PBS, 1% BSA and 0.2 U/l RNase Inhibitor). Nuclei were filtered twice in 40m strainers (Flowmi cell strainer, Sigma Aldrich). Isolated nuclei were stained with DAPI at concentrations suggested by the manufacturer and sorted by FACS (BD Influx BRV, Becton Dickinson Biosciences). Nuclei were collected, washed and resuspended in wash buffer. Nuclei were counted and appropriate volume for each sample was calculated for a target capture of 6000 nuclei. Samples were loaded onto a 10x Chromium controller using the Chromium Single Cell 3' Library & Gel Bead Kit v3 (10x Genomics). After droplet generation, Libraries were sequenced on an Illumina NovaSeq (10x specific protocol) with > 50, 000 reads per cell.