GSM2367498: MCF-7 siDDX54 replicate 2 IR 0h; Homo sapiens; RNA-Seq
Cells were harvested at different time periods after irradiation (0h, 1h, 2h and 6h) by removing the media and adding 1ml Trizol (Thermo Fisher) per well. RNA was then extracted by miRNAeasy kit (Qiagen) according to manufacturer's protocol. Next, 2 µg of total RNA were treated with 0.4 U Turbo DNase (Thermo Fisher) for 30 min at 37 ºC, phenol-chlorotorm extracted, ethanol-precipitated and resuspended in water. Equal amounts of total RNA (1 µg) and 2 µl of 1:100 dilution of ERCC Spike-in Control Mix 1 (Thermo Fisher) were mixed, adjusted to 50 µl final volume with water, and input into the Truseq unstranded mRNA kit v2 (Illumina) using 2 rounds of oligo-dT enrichment. Manufacturer's instructions were followed in all subsequent steps. cDNA libraries from different samples were multiplexed using Illumina RPI oligonucleotides and sequenced by multiplexing 6 samples per lane on a HiSeq 2000 instrument using 1x101+7 cycles. Illumina Truseq RNA v2 kit (unstranded)