title: GSM8439623: sham,replicate2,scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Single cells were collected into RPMI 1640 containing 5% FBS. Trypan blue exclusion confirmed the cell viability. Sorted cells were counted and concentration adjusted to 700 – 1,200 cells/μl. Then single-cell suspensions were loaded to a 10x Chromium to capture no more than 10,000 single cells by Chromium Single Cell 3' Reagent kits v.3 (10x Genomics). The cells were partitioned into Gel Beads in the Chromium instrument, where cell lysis and barcoded reverse transcription of RNA occurred. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM).The poly-A RNA from the cell lysate contained in every single GEM was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI), and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR, and the appropriate sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.