GSM7506011: Crlf3-mutant hippocampi; Mus musculus; RNA-Seq
Crlf3-mutant and wild type hippocampi, midbrains, and cortices were collected from four 4-week-old mice (4 hippocampi, 4 cortices, and 2 midbrains mice per genotype) and pooled to prepare single-cell isolations for each region, as previously reported . After collection, the brain tissues were dissociated using the neural dissociation kit (Miltenyi Biotec, 130-095-942) as per manufacturer's instructions followed by debris (Miltenyi Biotech, 130-107-677) and myelin (Miltenyi Biotec, 130-096-733) removal. The cells were then stained with 7-AAD and sorted using the MO-FLO. The live cells were collected and sent for sequencing. For all samples in the experiment, raw sequencing data was processed using the 10X Genomics Cell Ranger pipeline (version 6.1.1) to generate gene count matrices, and then aligned to the mouse reference genome (mm10-2020-A). cDNA was prepared after the GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps.  Purified cDNA was amplified for 11-16 cycles before being cleaned up using SPRIselect beads.  Samples were then run on a Bioanalyzer to determine the cDNA concentration.  Gene Expression libraries were prepared as recommended by the 10x Genomics Chromium Single Cell 5' Reagent Kit (v2 Chemistry Dual Index) user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration.  For sample preparation on the 10x Genomics platform, the Chromium Next GEM Single Cell 5' Kit v2, 16 rxns (PN-1000263), Chromium Next GEM Chip K Single Cell Kit, 48 rxns (PN-1000286) and Dual Index Kit TT Set A, 96 rxns (PN-1000215) were used.  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) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument.  Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow and a 151x10x10x151 sequencing recipe according to the manufacturer protocol.  A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression library.