GSM7006896: Tumor_infiltrated_Treg_WT; Mus musculus; RNA-Seq
Tumor tissue was resected and digested with enzyme. Lymphocytes were purified using percoll. Treg cells were purified by magnetic-activated cell sorting (MACS) positive selection. Tumor infiltrated treg of the WT and jmjd1c tregcKO mice were isolated by Fluorescence-activated cell sorting (FACS) according to the presence or absence of fluorence signal and analyzed using scRNAseq and then constructed library. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, cells 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 Ilumina 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 apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.