GSM8425876: CT26 xenografts, MCB-294, scRNAseq; Mus musculus; RNA-Seq
Tissues were transported in In sterile culture dish with 10 ml 1x Dulbecco's Phosphate-Buffered Saline (DPBS; Thermo Fisher, Cat. no. 14190144) on ice to remove the residual tissue storage solution, then minced on ice. We used dissociation enzyme 0.25% Trypsin (Thermo Fisher, Cat. no.25200-072) and 10 ug/mL lDNase I (Sigma, Cat. no. 11284932001) dissolved in PBS with 5% Fetal Bovine Serum (FBS; Thermo Fisher, Cat. no. SV30087.02) to digest the tissues, and then tissues were dissociated at 37 ℃ with a shaking speed of 50 r.p.m for about 40 min. We repeatedly collected the dissociated cells at interval of 20 min to increase cell yield and viability. Cell suspensions were filtered using a 40um nylon cell strainer and red blood cells were removed by 1 X Red Blood Cell Lysis Solution (ThermoFisher, Cat. no. 00-4333-57). Dissociated cells were washed with 1 x DPBS containing 2% FBS. Cells were stained with 0.4% Trypanblue (ThermoFisher,Cat.no.14190144) to check the viability on Countess® II Automated Cell Counter (ThermoFisher). The sample was then sent to Majorbio Bio-pharm Technology Co.,Ltd (Shanghai) for testing. Beads with unique molecular identifier (UMI) and cell barcodes were loaded close to saturation, so that each cell was paired with a bead in a Gel Beads-in-emulsion(GEM). After exposure to cell lysis buffer, polyadenylated RNA molecules hybridized to the beads. Beads were retrieved into a single tube for reverse transcription. On cDNA synthesis, each cDNA molecule was tagged on the 5' end (that is, the 3' end of a messenger RNA transcript) with UMI and cell label indicating its cell of origin. Briefly, 10×beads that were then subject to second-strand cDNA synthesis, adaptor ligation, and universal amplification. Sequencing libraries were prepared using randomly interrupted whole-transcriptome amplification products to enrich the 3' end of the transcripts linked with the cell barcode and UMI. All the remaining procedures including the library construction were performed according to the standard manufacturer's protocol (Chromium Single Cell 3ʹv3.1). Sequencing libraries were quantified using a High Sensitivity DNA Chip(Agilent) on a Bioanalyzer 2100 and the Qubit High Sensitivity DNA Assay (Thermo Fisher Scientific). The sequencing libraries were performed on DNBSEQ-T7 platform (PE150) using DNBSEQ-T7RS Reagent Kit (FCLPE150) version 3.0. The sequencing and bioinformatic analysis were performed on platform of Majorbio Co.,Ltd (Shanghai,China).