title: GSM4827299: Nrf2 cKO rep2; Mus musculus; RNA-Seq
library_construction_protocol: Microdissection of a ~2.5mm concentric ring around the 10mm wound was carried out using a 15mm-diameter punch biopsy. Wounded skin was harvested at indicated time points and digested in 10u/mL Dispase (Thermo Fisher, 17105041) and 5ug/mL Accutase (Bio-Legend, 42301) before passing the suspension through a 70um pore cell trainer. Lineage-positive cells and dead cells/debris were excluded from the cell suspension utilizing the MACS Lineage Depletion Kit (Miltenyi, 130-090-858) and MACS Dead Cell Removal Kit (Miltenyi, 130-090-101), respectively, following the manufacturer's instructions. Cell number and viability was assessed using trypan blue and a hemocytometer. Dissociated single cells were suspended in 5% FBS in 1x PBS and incubated with various antibody combinations for multi-parameter flow acquisition and analysis. Live keratinocytes were sorted using antibodies directed against the following surface makers: Lin- (CD5-, CD45R-, CD11b-, Cr-1-, 7-4-, Ter-119-) (Miltenyi, 130-090-858), CD49f+ (Bio-Legend, 313610) using a SY3200 4 laser/10 color cell sorter. For RNA-seq, cDNA library preparation used optimal quality RNA (RIN>9). Full length cDNA library amplification was performed as previously described. Briefly, total RNA was reverse-transcribed and the resulting cDNA was pre-amplified using the Nugen Trio RNAseq Kit (Nugen Inc.). Paired-end 50 (PE50) sequencing was performed on the Illumina HiSeq 4000 (Illumina).