RNA-seq of Mus musculus skin tissue
Total RNA was isolated using the Trizol Reagent (Invitrogen Life Technologies), after which the concentration, quality and integrity were determined using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were used as input material for the RNA sample preparations. Sequencing libraries were generated according to the following steps: firstly, mRNA was purified from total RNA using poly - T oligo - attached magnetic beads; fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer; first strand cDNA was synthesized using random oligonucleotides and Super Script II; second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H; remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed; after adenylation of the 3 ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization; to select cDNA fragments of the preferred 400 - 500 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, CA, USA); DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction; products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on NovaSeq 6000 platform (Illumina).