GSM8188184: Young_GIA; Mus musculus; RNA-Seq
Inguinal lymph nodes (LNs) were harvested from young (8 weeks) and old (14-15 months) DBA/1 mice 14 days after GIA induction (GIA groups) or PBS injection (control groups). The LNs were homogenized and filtered 40um cell strainer and washed with PBS. All cells from LNs were stained with Pacific blue-conjugated anti-mouse CD4 antibody and CD4+ T cells were sorted on a Moflo XDP cell sorter (Beckman Coulter) . CD4+ T cells were collected equally from four mice in each of the four groups, each with 10,000 cells. CD4+ T cells were washed with PBS and resuspended in 0.04% w/v BSA. Library was performed according to the manufacter's instructions (Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1, 10x Genomics). Briefly, CD4+ T 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.