title: GSM8073730: Hypoxia Nnt-wt mouseID=HMW2; Mus musculus; RNA-Seq
library_construction_protocol: Directly after normoxia/hypoxia exposure, mice were killed by dicapitation. Gastrocnemius was isolated, snap frozen in liquid nitrogen and stored at -80°C until further analysis. Total RNA was extracted by grinding frozen muscle tissue and dissolving approximately 15 mg in 1 ml Trizol. Further RNA isolation was performed according to manufacture's  instructions (Thermo Fisher). RNA concentration was measured using NanoDrop one spectrophotometer (Thermo Fisher). RNA integrity was measured using RNA Screentape on the 2200 Tapestation (Agilent). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using dUTP for directional library. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification(In-house kit: Novogene NGS Stranded RNA Library Prep Set (PT044)). The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms (NovaSeq 6000 platform using S4 flow cells), according to effective library concentration and data amount.