title: GSM7847866: SIY-OVAII DLN; Mus musculus; RNA-Seq
library_construction_protocol: Tumor bearing mice were sacrificed, tumor and draining lymph node was isolated and placed in ice-cold phosphate-buffered saline (PBS). For draining lymph node, the tissue was minced and dissociated with 0.25% trypsin in a centrifuge tube for 30 min at 28 °C, then the sample was filtered using a 70 µm strainer to obtain a single-cell suspension. for CD8 tumor infiltrating T cell, the tumor was digested by collagenase IV for 40 min, single cell suspension of tumor cell was stain with anti-CD8 and anti-CD3 antibody, CD8 tumor infiltrating T cell was sorted by flow cytometry (Melody,BD). Using single-cell 5 'Library and Gel Bead Kit V3 (10× Genomics) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074), the cell suspension (10000 living cells per microliter determined by Count Star) was loaded onto the Chromium single cell controller (10× Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. In short, single cells were suspended in PBS containing 0.04% bovine serum albumin (BSA). Approximately 10000 cells were added to each channel, and the target cell recovered was estimated to be approximately 3000 cells. Captured cells were lysed, and the released RNAs were barcoded through reverse transcription in individual GEMs. Reverse transcription was performed on an S1000TM Touch Thermal Cycler (Bio Rad) at 53 °C for 45 min followed by 85 °C for 5 min, and incubated at 4 °C. The complementary DNA (cDNA) was generated and then amplified, and the quality was assessed using an Agilent 4200. According to the manufacturer's instruction, scRNA-seq libraries were constructed using Single Cell 5' Library and Gel Bead Kit V3. The libraries were finally sequenced using an Illumina Nextseq 2000 sequencer with a sequencing depth of at least 100,000 reads per cell with a pair-end 150 bp reading strategy. RNA libraries were prepared with Chromium Single cell 5' Reagent v3 kits according to the manufacturer's protocol