mRNA-seq of NRK cell line: nc transfection 32h replicate 2
Total RNAs of cells were extracted using the TRIzol reagent (Thermo Fisher) and chloroform.Sequencing libraries were generated using NEBNext_ UltraTM Directional RNA Library Prep Kit for Illumina_ (NEB, USA) following manufacturerĄŻs recommendations. Briefly, 10~20 ŚĚg Poly(A)+ RNA selected on oligo-dT25DynaBeads (Thermo Fisher) and fragmented at 70ĄăC for 15 min in fragmentation reagent (Thermo Fisher). Fragmented RNAs within 40~80 nt were 3ĄŻ-dephosphorylated, ligated with 3ĄŻ-adaptor and purified on a 15% TBE-Urea gel. The 3ĄŻ-ligated RNAs were then 5ĄŻ-phosphorylated, ligated with 5ĄŻ-adaptor and reverse-transcribed. All cDNAs were amplified by 12 PCR cycles and the products within the correct size ranges were collected from 20% TBE gels for quality tests (Fragment Analyzer, Agilent Technologies) and sequenced on Illumina Hiseq platform and single-end reads were generated.