NextSeq 550 sequencing; Single cell RNA-sequencing of non-erythrocyte splenocytes from control and social defeated C57BL6 mice
Single cell RNA-sequencing of non-erythrocyte splenocytes from control and social defeated C57BL6 mice
Mouse spleen isolated, physically disrupted with ground glass, filtered through 70uM mesh, erythrocytes lysed via red blood cell lysis buffer, filtered through 70uM mesh. Cell suspensions were evaluated by light microscopy for debris and viability, and were counted using a hemocytometer. Cell concentrations were 932 and 888 live cells / ul respectfully. Targeting approximately 2400 single cells per sample, single cells were captured, lysed, and RNA was reverse transcribed and barcoded using a 10x Genomics Chromium instrument and  Chromium Single Cell 3' Reagent Kits v2 reagents. (10x Genomics, Pleasonton, CA). To construct Illumina compatible sequencing libraries the cDNA is fragmented, A-tail repaired and a double-sided bead cleanup is performed. Adapters are ligated to the cDNA fragments and the fragments are PCR amplified using unique sample index primers per manufacturer's recommendations. Libaries were quantified by qPCR using the KAPA Library Quant Kit (Illumina) from KAPA Biosystems (Roche, Pleasonton, CA).
Experimental Factor: environmental stress
control