KEAP1_NRF2_19RK57_5_12_S20
RNA was extracted from 15 mg grinded hearts tissues. TissueLyser LT (Qiagen, Germany) was used to homogenize the tissue in TRIzol for 2 min at 50Hz. After obtaining the RNA pellet, the pellet was washed twice with 80% ethanol and dissolved in 90 L of RNase free water. Residual gDNA was then digested with the DNase Max (Qiagen) and the big-RNA fraction (>200nt) was recovered with RNeasy MinElute Cleanup Kit (Qiagen) following the manufacturers instructions. The extracted RNA was quantified with Qubit RNA BR assay (Invitrogen) and the quality of RNA was assessed by Fragment Analyzer standard sensitivity RNA kit (Agilent Technologies, USA).Prior to RNA-seq, integrity and quantity of the RNA were assessed by Fragment Analyzer Standard Sensitivity RNA kit (Agilent Technologies, USA). All samples had an RNA integrity number >8.7 and were deemed of sufficient quality for mRNA-seq analysis. 1000 ng of total RNA was used as input to each mRNA-seq library. KAPA mRNA HyperPrep kit (Roche, Switzerland) was used for reverse transcription, generation of double stranded cDNA and subsequent library preparation and indexing according to manufacturers instructions. Quality and quantity of libraries was assessed by Fragment Analyzer standard sensitivity NGS kit (Agilent Technologies, USA). Indexed libraries were pooled in equimolar ratios. The sample pool was quantified with a Qubit Fluorometer (ThermoFisher Scientific, USA) using the dsDNA HS kit (ThermoFisher Scientific, USA), further diluted, and sequenced to >15M paired reads/sample on NextSeq500/550 (Illumina, USA) with PE 75 base pair (bp) read length setting.