title: GSM8153075: sample_2; Mus musculus; RNA-Seq
library_construction_protocol: Freshly collected RENCA tumors from BALB/c mice were transferred to a Petri dish (Petri dish was placed on wet ice) pre-spiked with 1×PBS (without RNase and Ca, Mg ions) and washed with 1XPBS to remove blood stains, grease and other adherents from the tissue surface. The tissue was then cut into 0.5 mm2 pieces and washed again with 1×PBS. The washed pieces were added with dissociation solvent(0.35% collagenase IV5, 2 mg/ml papain, 120 Units/ml DNase I) and reacted for 20 min at 37°C in a water bath shaker (100 rpm). PBS (containing 10% fetal bovine serum) was used to terminate the dissociation. The cells were repeatedly puffed 5-10 times with a pipette gun (gentle handling to prevent cell death due to shear force). The cell suspension was filtered through a 70-30um cell sieve and subsequently centrifuged at 300g for 5 min at 4°C. After centrifugation, the cell sediment was collected and the cells were resuspended by adding 100ul of 1× PBS (0.04% BSA) solution. To remove erythrocytes, 1 ml of 1X erythrocyte lysis solution (MACS 130-094-183, 10×) was added and reacted at room temperature or on wet ice for 2-10 min. After lysis, the cells were centrifuged at 300g for 5 min at 4°C, and the cell precipitate was collected after centrifugation. Add 100μl of Dead Cell Removal MicroBeads (MACS130-090-101), mix well and incubate for 15min at room temperature. At the end of incubation, Binding Buffer was added, after MS Columns (130-042-201) to remove reagents, and dead cells.Cell precipitates were collected by centrifugation at 300g for 5min at 4°C. Subsequently 1×PBS (0.04% BSA) was used to resuspend the cell precipitate and centrifuged at 4°C 300g for 5min(repeated twice). The candidate cells were obtained after tissue dissociation,lysis of erythrocytes and removal of dead cells, and 100 μl of 1×PBS (0.04% BSA) was added to form a cell suspension. The cell activity was detected by trypan blue staining method, and the cell activity was required to be >85%. The number of cells was counted using a hemocytometer or Countess II Automated Cell Counter, and the cell concentration was 700-1200 cells/μl. Libraries were generated using the 10X Genomics Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3. Briefly, cells were loaded into the Chromium Controller (10x Genomics) where they are partitioned into nanoliter-scale Gel Beads-in-emulsion. Reverse transcription was performed, and the resulting cDNA was amplified following the manufacturer's instructions. The full-length amplified cDNA was used to generate gene expression libraries by enzymatic fragmentation, end-repair, a-tailing, adapter ligation, and PCR to add Illumina compatible sequencing adapters. The resulting libraries were evaluated on D1000 screentape using a TapeStation 4200, and quantitated using Kapa Biosystems qPCR quantitation kit for Illumina. Final libraries were then pooled, denatured, and diluted to 300 p.m. with 1% PhiX control library added. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run,150bp) by LC-Bio Technology co.ltd., (HangZhou,China) at a minimum depth of 20,000 reads per cell.