title: GSM7422897: wt littermate, pooled CD45+ cells; Mus musculus; RNA-Seq
library_construction_protocol: brain tissues were collected inot RPMI media and enzymatically dissociated using collagenase/dispase and DNAase (1h/37C). Following this, the cellular suspension was filtered (70uM) and then separated on a 70/30 Percoll gradient where leukocytes were collected at tht interphase. CD45+ cells were collected by flowcyotmetry (FACSARIA II, Becton-Dickinson). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). CD4+ cells were resuspended in a master mix that was 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.