GSM8374448: choroid plexus CD45+ cells, LCMV-1; Mus musculus; RNA-Seq
Choroid plexus CD45+ live cells were sorted and resuspended in 40 μl of PBS + 0.04% nuclease-free BSA (Sigma). The entire volume was loaded into the 10x Chromium chip. 5 mice from the same experimental group were pooled for each individual sample. Single-cell gene expression libraries and sequencing were generated using the Next GEM Single Cell 3' Reagents Kit from 10x Genomics. The Chromium Next GEM Single Cell 3' Kit v3.1, 16 rxns (PN-1000268), Chromium Next GEM Chip G Single Cell Kit, 48 rxns (PN-1000120), and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used. Single cells partitioned into Gel-bead-in-Emulsions (GEMs) droplets underwent retro-transcriptase reaction, and cDNA received a unique 12-nucleotide barcode and unique molecular identifier (UMI). cDNA was amplified for 11 cycles before being purified using SPRIselect beads. GEX libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 3' Reagent Kits User Guide (v3.1 Chemistry Dual Index) with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche). Normalized cDNA libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 28x10x10x150 sequencing recipe according to the manufacturer's protocol (Illumina). A median sequencing depth of 50,000 reads/cell was targeted for each gene expression library.