GSM7156164: IEL_gdT; Mus musculus; RNA-Seq
Mouse tumor tissues were cut up into fragments that were smaller than 0.05 mm in length and digested. The cells were washed with incomplete medium by centrifugation at 400 g for 10 minutes, and then separated by Percoll density gradient centrifugation at 800 × g for 25 min at room temperature. A mixed cell suspension was obtained from the 40%/70% Percoll interface and washed. Library was performed according to the manufacturer's instructions (single cell 3' v3 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 retrotranscripted 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 appropriately 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.