title: GSM8761585: CM272 and anti-PD-1, sample 2; Mus musculus; RNA-Seq
library_construction_protocol: On day 15, tumors were harvested and processed under sterile conditions. Single-cell suspensions of tumors were obtained by mechanical and enzymatic dissociation using 1 mg/mL collagenase D and 50 µg/mL DNase I at 37°C for 30 minutes. EDTA (6 µM) was then used to block collagenase and DNase I activities. Erythrocytes were lysed, and cells were resuspended in 35% Percoll solution and centrifuged at 2,000 rpm for 15 minutes without brake. The supernatant was discarded and cells were preincubated with Fc block for 15 minutes at 4ºC, and then labeled with fluorochrome-conjugated antibodies against CD45, CD11b and Ly6G. Dead cells were labeled with SYTOX Blue. CD45+ and double CD11b-/Ly6G- cells were sorted. The transcriptome of the cells was sequenced using Single Cell 3' Reagent Kits v3.1 (10X Genomics). Briefly, 20,000 cells (except for sample Triple_2, where only 5,840 cells could be obtained) were loaded at a concentration of 1,000 cells/µL on a Chromium X instrument (10X Genomics) to capture single cells in gel bead-in-emulsions (GEMs). In this step, each cell was encapsulated with primers containing a fixed Illumina Read 1 sequence, a cell-identifying 16 nt 10X barcode, a 12 nt unique molecular identifier (UMI), and a poly-dT sequence. After cell lysis, reverse transcription yielded full-length barcoded cDNA. This cDNA was then released from the GEMs, amplified by PCR and purified using magnetic beads (SPRIselect, Beckman Coulter). Enzymatic fragmentation and size selection were used to optimize cDNA size prior to library construction. Fragmented cDNA was end-repaired, A-tailed and ligated to Illumina adaptors. A final PCR amplification with barcoded primers allowed for sample indexing. Library quality control and quantification were performed using the Qubit 3.0 Fluorometer (Life Technologies) and the 4200 TapeStation System (Agilent), respectively. Sequencing was performed on NextSeq2000 and NovaSeq6000 instruments (Illumina) aiming for at least 20,000 reads/cell.