GSM8530981: A549 cells, shTRIM22, 3 days after ionizing radiation1; Homo sapiens; RNA-Seq
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.3 ug of total RNA was used was used for the construction of sequencing libraries Briefly, mRNA was purified from total RNA using poly-T oligo-attached magneticbeads. Fragmentation was carried out using divalent cations under elevated temperature inFirst Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized usingrandom hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strandcDNA synthesis was subsequently performed using DNA Polymerase I and RNase H.Remaining overhangs were converted into blunt ends via exonuclease/polymerase activitiesAfter adenylation of 3'ends of DNA fragments, Adaptor with hairpin loop structure wereligated to prepare for hybridization.In order to select cDNA fragments of preferentially370~420 bp in length, the library fragments were purified with AMPure XP system(Beckman Coulter, Beverly, USA). Then PCR was performed with Phusion High-FidelityDNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products werepurified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer2100 system.