title: GSM8951716: Control 1; Mus musculus; RNA-Seq
library_construction_protocol: Mice were transcardially perfused with cold PBS and cortical regions were quickly dissected and enzymatically digestssss(Miltenyi Biotec, 130-092-628) in the gentleMACS Octo Dissociator with heaters (Miltenyi Biotec) using program 37C_NTDK_1. The myelin was then removed by magnetic bead separation using Myelin Removal Beads II (Miltenyi Biotec, 130-096-733) followed by the red blood cell lysis (Miltenyi Biotec ,130-094-183). Cell suspension was then followed by dead cell removal (Miltenyi Biotec 130-090-101;). The final cell pellet was suspended in Ca/Mg-free PBS with 0.5% BSA, and immediately submitted to the Genome Analysis Core for Single Cell partitioning. The cells were counted and measured for viability using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter). The barcoded Gel Beads were thawed from -80 °C and the cDNA master mix was prepared according to the manufacture's instruction for Chromium Next GEM Single Cell 3' Kit v3.1 (10x Genomics).  Based on the desired number of cells to be captured for each sample, a volume of live cells was mixed with the cDNA master mix.  A per sample concentration of 500,000 cells per milliliter or better is required for the standard targeted cell recovery of up to 10,000 cells. The stock concentration requirements would not change for higher cell recovery numbers. The cell suspension and master mix, thawed Gel Beads and partitioning oil were added to a Chromium Next GEM G chip. The filled chip was loaded into the Chromium Controller, where each sample was processed and the individual cells within the sample were partitioned into uniquely labeled GEMs (Gel Beads-In-Emulsion). The GEMs were collected from the chip and taken to the bench for reverse transcription, GEM dissolution, and cDNA clean-up. The resulting cDNA contains a pool of uniquely barcoded molecules. A portion of the cleaned and measured pooled cDNA continues to library construction, where standard Illumina sequencing primers and a unique sample index (Dual Index Kit TT-Set A; 10x Genomics) were added to each cDNA pool, creating gene expression libraries.  All cDNA pools and resulting libraries are measured using Qubit High Sensitivity assays (Thermo Fisher Scientific) and Agilent Bioanalyzer High Sensitivity chips (Agilent).