title: GSM7680550: Nrf2Act-Hep A8; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated as above together with RNase-Free DNase kit (Qiagen, #79254) to obtain pure RNA. RNA degradation and contamination were monitored on 1% agarose gels and RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA integrity and quantitation were assessed with the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). For library preparation, a total of 1 μg RNA per sample was used as input for RNA sample preparations. Sequencing libraries were generated with NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following the manufacturer's protocols and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and fragmented using divalent cations under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer (5×). First strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA synthesis was performed with DNA polymerase Ⅰ and RNase H. Remaining overhangs were converted into blunt ends by exonuclease/polymerase activities. After 3' end adenylation of DNA fragments, NEBNext adaptors with hairpin loop structures were ligated to prepare for hybridization. Library fragments were then purified with AMPpure XP system (Beckman Coulter, Beverly, USA) and 3 μl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNAs at 37℃ for 15 min followed by 5 min at 95℃. PCR was then performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Library quality was assessed on the Agilent Bioanalyzer 2100 system.