title: GSM6886319: Surgery alone, adjacent normal sample, scRNAseq (S9478N); Homo sapiens; RNA-Seq
library_construction_protocol: The samples for scRNA-seq were washed with phosphate-buffered saline (PBS; Thermo Fisher Scientific, Waltham, MA, USA), placed on ice, cut into small pieces (< 2 mm3) and transferred to 5 mL Roswell Park Memorial Institute 1640 (RPMI; Thermo Fisher Scientific) containing collagenase IV (1 μg/mL), collagenase I (2 μg/mL) and DNase (1 μg/mL) (Thermo Fisher Scientific). The samples were incubated for 40 min at 37 °C with manual shaking every 5 min and then filtered through both 70-µm and 40-µm nylon mesh (Thermo Fisher Scientific). Following centrifugation (300 × g, 4 °C, and 5 min), the samples were then resuspended in PBS containing 0.04% bovine serum albumin (BSA; Sigma). Next, the cells were counted and assessed for viability using Trypan blue staining with a hemocytometer. During the dissociation procedure, the cells were kept on ice whenever possible, and the entire procedure was completed in < 90 min (generally ~70 min) to avoid the dissociation-associated artifacts recently described. A positive signal for dissociation signatures that reflects dissociation-associated changes in gene expression was obtained in < 1% of the cells. Droplet-based scRNA-seq, a chromium single-cell 3′-library, was constructed using the Chromium Single-cell 3′-Library, Gel Bead & Multiplex Kit and Chip Kit (10× Genomics, Pleasanton, CA, USA) according to the manufacturer's instructions. Cell suspensions were loaded onto a chromium single-cell chip along with reverse transcription (RT) master mix and single-cell 3′-gel beads, aiming for 2,000–8,000 single cells per reaction. The samples were processed using 10× Genomics V3 barcoding chemistry kits (Supplementary information, Table S2). Following cell lysis, first-strand cDNA synthesis and amplification were carried out according to the manufacturer's instructions with cDNA amplification set for 12 cycles.