title: GSM2262572: SuRE K562 cDNA, Biorep1, PCRrep2; Homo sapiens; OTHER
library_construction_protocol: RNA was isolated using Trisure (#BIO-38032; Bioline) and polyA RNA was purified using Oligotex from Qiagen (#70022; Qiagen). PolyA RNA was divided into 10 µl reactions containing 500 ng RNA and DNase treated with  10 units for 30 minutes (#04716728001; Roche) and DNase was inactivated by addition of 1µl 25mM EDTA and incubation at 70ºC for 10 minutes. Next, cDNA was produced by first adding 1 µl of 10 mM gene specific primer targeting the GFP ORF (247JvA) and 1 µl dNTP (10mM each) and incubating for 5 minutes at 65°C. Then 4 µl of RT buffer, 20 units RNase inhibitor (#EO0381; ThermoFisher Scientific), 200 units of  Maxima reverse transcriptase (#EP0743; ThermoFisher Scientific)  and 2.5 µl water was added and the reactionmix was incubated for 30 minutes at 50°C followed by heat-inactivation at 85° for 5 minutes. Per biological replicate, 20-30 reactions were done in parallel. Each 20 µl reaction was then PCR amplified (1’ 96 °C, 20x(15’’ 96 °C, 15’’ 60 °C, 15’’ 72 °C)) in a 100 µl reaction with MyTaq™ Red Mix and primers 151AR (containing the S1 and p5 adapter) and (index variants of) 211JvA (containing the S2, index and p7 adapter) for 21 cycles. Reactions were then pooled and 500 µl was purified using a PCR purification kit (#BIO-52060) and then size-selected using e-gel (#G6400EU; Invitrogen) and subjected to single read 50 bp high throughput sequencing on an Illumina HiSeq2000 or HiSeq2500.
attributes.tag: GEO Accession
attributes.value: GSM2262572