title: HiSeq X Ten paired end sequencing; Transcriptome and translatome of cardiomyocytes exposed to hypoxia in vivo and in vitro
library_construction_protocol: adult male SD rats (6-8-week old, 337.58 ± 29.84 g) were subjected to acute normobaric hypoxia at 10% oxygen for 10 and 30 min. Rat H9C2 cardiomyocytes were treated with the culture condition (1% O2, 94% N2, and 5% CO2) for 8 hr and 24 hr. 1 mg Rat cardiomyocytes in border zone of infarct and 10E6 H9C2 cells with mild and severe hypoxia were extracted RNA using Trizol for the subsequent RNA-seq and Ribo-seq. For Ribo-seq, one additional step was required for RNA isolation. After RNA was released from cells, 15-40% linear sucrose-gradient centrifugation at 38,000 rpm for 2.5 h at 4 oC by a SW41 Ti rotor (Beckman Coulter, Brea, CA, USA). Fractionation with the peaks of A254 absorbing material followed by free mRNA, 40S, 60S and 80S successively were polysomal peaks. Polysomal peaks were harvested to extract RNA, which represented polysome profiles, namely translatome.2 μg RNA of each sample was used for library preparation by NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) following manufacturer's recommendations and were sequenced on an Illumina Hiseq platform.