GSM7925501: Hippocampus, Trem2 WT, rep 1; Mus musculus; RNA-Seq
Freshly collected hippocampi from Trem2 P1 pups were dissected into ice-cold HBSSwo (Mg2+/Ca2+ free supplemented with 10mM HEPES, pH 7.3) to remove fibers. Single cell dissociation was performed by incubation of tissue with papain dissociation solution according to the manufactured instructions (Papain Dissociation System Worthington #LK003150) in a shaking incubator for 30 minutes at 37°C. After incubation the tissue was mechanically dissociated into single cell suspension and the cells were washed and re-suspended in PBS supplemented with 0.02% w/v BSA for cell counting and viability assessment. Library was performed according to the manufacter's instructions (single cell 3' v3.1 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, single-end constructs with P5 and P7 sequences and a sample index were added.