title: GSM6918973: WT mouse 2 scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Paw skin was dissected, dissassociated into a single cell suspension, and washed. Sample KOD11 was passed through a ficoll suspension to remove RBCs. Single cell library preparation and sequencing were done by Singulomics Corporation (https://singulomics.com/, Bronx, NY). Fresh, viable single cell suspensions were washed and resuspended in cell culture media with 0.04% BSA, and counted. Viable cell suspensions were loaded into the Chromium Controller (10x Genomics, Pleasanton, CA) to generate gel beads-in-emulsion (GEM) with each GEM containing a single cell as well as barcoded oligonucleotides. Next, the GEMs were placed in the SimpliAmp 96-well Thermal Cycler (Thermo Fisher Scentific, Wilmington, DE) and reverse transcription was performed in each GEM (GEM-RT). After the reaction, the complementary cDNA was amplified and cleaned using Silane DynaBeads (10X Genomics, Pleasanton, CA) and the SPRIselect Reagent kit (Beckman Coulter, Indianapolis, IN). Amplified full-length cDNAs from poly-adenylated mRNA were then used to generate 5ʹ Gene Expression library (Chromium Next GEM Single Cell 5' Reagent Kits v2 - Dual Index), following the manufacturer's instructions (10x Genomics, Pleasanton, CA). Amplified cDNAs and the libraries were measured by Qubit dsDNA HS assay (Thermo Fisher Scientific, Wilmington, DE) and quality assessed by BioAnalyzer (Agilent Technologies, Santa Clara, CA). Libraries were sequenced on a NovaSeq 6000 instrument (Illumina, San Diego, CA, United States), and reads were subsequently processed using 10x Genomics Cell Ranger analytical pipeline (v6.0.1) and mouse reference genome mm10. Dataset aggregation was performed using the cellranger aggr function normalizing for the total number of confidently mapped reads across libraries.