GSM8587147: sc189_1; Mus musculus; RNA-Seq
Single-cell suspensions from tumors taken 6 days after treatment were stained with Zombie Nir and CD45. Viable CD45+ cells were sorted in a MoFlo Astrios-EQ cell sorter (Beckman Coulter). Droplet-based isolation of single cells was performed with the Chromium Controller (10x Genomics). 3′ sequencing libraries was performed per the manufacturer's instructions (10x Genomics). Briefly, 17000 to 20000 cells were loaded at a concentration of 1000 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. Upon cell lysis, reverse transcription yielded full-length, barcoded cDNA. This cDNA was then released from the GEMs, PCR-amplified and purified with magnetic beads (SPRIselect, Beckman Coulter). Enzymatic Fragmentation and Size Selection was used to optimize cDNA size prior to library construction. Fragmented cDNA was then end-repaired, A-tailed and ligated to Illumina adaptors. A final PCR-amplification with barcoded primers allowed sample indexing. Library quality control and quantification was performed using Qubit 3.0 Fluorometer (Life Technologies) and Agilent's 4200 TapeStation System (Agilent), respectively. Sequencing was performed in a NextSeq2000 (Illumina) using paired-end 91-base pair reads.