Truseq standard mrna library kit

RNA was isolated from either whole skin biopsies, flash frozen and then homogenized (in the case of Stat3 cKO skin for cytokines) with a Bessman Tissue Pulverier (Spectrum TM) or FASC-purified EpdSCs using Direct-zol RNA Miniprep Kit (Zymo Research) as per manufacturer’s instructions. Equal amounts of RNA were reverse-transcribed using the superscript VILO cDNA synthesis kit (Invitrogen). For quantitative PCR, biological replicates represent the average of three technical replicates per individual animal. cDNAs from each sample were normalized using primers against Actb. For a complete list of qPCR primers, see Supplementary Table 2.
For analysis of RNA-Seq from Naik et al, FASTQs can be accessed from GSE92967 (Naik et al., 2017 (link)). Reads from D6 Ctrl, D6 IMQ, D30 Ctrl, D30 PI, Ctrl 6 hr post-wound, and PI 6hr post-wound were reanalyzed with Salmon (v 1.4.0) using the Gencode Release M25 (GRCm38.p6) transcriptome (Patro et al., 2017 (link)). For RNA-seq of 6hr post-TPA treatment, libraries were prepared using Illumina TruSeq standard mRNA library kit and sequenced on Illumina NovaSeq 6000. Analysis was performed as described above.