title: GSM6700860: Primary tumor-EO771-HEVM3, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Briefly, primary tumors and paired lymph nodes from six C57BL/6J mice were collected, washed with Hanks Balanced Salt Solution three times and minced into small pieces using surgical scissors. The tumor tissues were digested with DMEM medium containing 2 mg/ml collagenase I (Worthington, #LS004196), 2 mg/ml collagenase III (Worthington, #LS004182) and 2 mg/mL hyaluronidase (Sigma Aldrich, #H3506) for 2 h at 37 °C under constant shaking. The lymph node tissues were digested with RPMI-1640 medium containing 0.2 mg/ml collagenase P (Sigma Aldrich, #11213857001), 0.8 mg/ml dispase Ⅱ (Sigma Aldrich, #D4693) and 0.01 mg/ml DNase (Sigma Aldrich, #DN25) for 1 h at 37 °C under constant shaking. FBS (final concentration 30%) was added to stop the digestion. Dissociated cell suspensions were then passed through 100 μM and 40 μM filter. Cells were pelleted at 250 g for 5 min, and treated with 2ml of 1× RBC lysis buffer (Biolegend, #420301) on ice for 10 min. The washing buffer was then added and the cells were centrifuged at 1000 rpm for 5 min and was resuspended in PBS. The single-cell suspension was converted to barcoded scRNA-seq libraries using the Chromium Single Cell 3' Library, Gel Bead & Multiplex Kit and Chip Kit (10× Genomics) according to the manufacturer's protocol. Briefly, the single-cell suspension was loaded into each reaction. The RNA transcripts were uniquely barcoded and reverse-transcribed. Complementary DNA (cDNA) was generated, amplified, fragmented, end repaired, and ligated with adaptors to generate sequencing libraries.