title: GSM8545019: Mouse Skin SCA1+ Fibroblasts, 1.5dpw, 3; Mus musculus; RNA-Seq
library_construction_protocol: Unwounded and wounded skin samples were minced with scissors and digested in a liberase digestion solution. Cells were passed through 70μm and 40μm strainers. Cells were incubated with an anti-CD45-APC-Cy7, anti-CD31-APC-Cy7, and anti-SCA1-BV650 antibodies and stained with SYTOX Blue viability dye. Live, CD45-, CD31-, SCA1+ cells were sorted from each sample on a Sony SH800 instrument. RNA extraction was immediately performed using the Agilent Absolutely RNA Nanoprep Kit. RNA Integrity Number scores for all 12 samples were assessed using an Agilent Bioanalyzer RNA 6000 Pico kit (p/n 5067-1513), producing measurements suitable for library preparation using the Takara Bio USA, Inc SMART-Seq® v4 Ultra® Low Input RNA Kit (p/n 634888). Total RNA input ranged from 76-789 ng (average 375 ng), with an minimum RIN score of 7. General requirements and recommendations were followed for full-length cDNA amplification and purification. cDNA were quantified and assessed using an Agilent Bioanalyzer High Sensitivity DNA kit (p/n 5067-4626) and a Thermo Fisher Qubit 3.0 High Sensitivity DNA kit (p/n Q33230). cDNA samples were diluted and 150 pg input was used as input, per sample, in the Illumina Nextera XT Library Preparation kit (p/n FC-131-1024), per recommendations from the Takara SMART-Seq® v4 Ultra® Low Input RNA Kit user manual. Nextera XT libraries were quantified and assessed using an Agilent Bioanalyzer High Sensitivity DNA kit and a Thermo Fisher Qubit 3.0 High Sensitivity DNA kit. Libraries were pooled and sequenced using an Illumina NextSeq 500 High-Output 150 cycle v2.5 kit (p/n 20024907), generating 470 million paired-end reads. FastQ files were generated and demultiplexed, with even read depth across the twelve samples.