GSM5702534: Fractionally Irradiated to 20 Gray Replicate 3; Homo sapiens; RNA-Seq
Three independent replicates each of exponentially growing PAR, AMC and FIR20 cells were seeded in 25 cm flasks and incubated for 48 h followed by RNA extraction. Cell pellets were collected by trypsinization, snap frozen and stored at -80oC until further use. Total RNA was extracted from the cell pellets using RNeasy Mini Kit (Qiagen, Venlo, The Netherlands) as described by the manufacturer. During the extraction procedure, genomic DNA was removed using a genomic DNA eliminator spin column (Qiagen). Sample RNA concentrations were measured and assessed for the presence of contaminants using a NanoDrop ND-2000 spectrophotometer (Thermo Scientific, USA) and the DropSense™ 16 spectrometer (Trinean, Ghent, Belgium). RNA integrity was subsequently assessed using Agilent 2100 bioanalyzer (Agilent Technologies, Inc Palo Alto, CA, USA). Total RNA was depleted of ribosomal RNA (rRNA) using the Ribo-Zero HMR kit (Illumina, USA) according to manufacturer instructions. The rRNA-depleted RNA was then purified using 2x RNAClean XP beads (Beckman Coulter) followed by first and second-strand synthesis using NEBNext, New England Biolabs reagents. The generated cDNA was purified with 1.8x SPRIselect Beads (Beckman Coulter). Endprep reaction was performed following the manufacturer's protocol for NEBNext Ultra II End Prep Reaction for the rest of the procedure. Adaptor ligation was then performed using NEBNext Ultra II Ligation protocols (New England Biolabs) and the ligated product was purified by SPRIselect Beads (Beckman Coulter) followed by elution in nuclease-free water. PCR was carried out using NEBNext Ultra II Q5 Master Mix, and primers and the final library was subsequently purified with SPRIselect Beads