title: GSM1142038: HEK293 cells transfected with pCag-EGFP plasmid.; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using RNAzol RT (Molecular Research Center). Further sample processing and SOLiD sequencing was performed by Seqomics Ltd. (Hungary). Briefly, samples were depleted of ribosomal RNA using RiboMinus rRNA Removal Kit, eluted twice int Sample processing and SOLiD sequencing was performed by Seqomics Ltd. (Hungary). Briefly, samples were depleted of ribosomal RNA using RiboMinus rRNA Removal Kit, eluted twice into 15 ul and treated with DNase I (5 ul 10x DNase I buffer, 2.5 U DNase I and water up to 50 ul reaction) 20 min at 37°C. Samples were ethanol-precipitated, eluted into 10 ul TE buffer and fragmented by RNase III treatment (1 ul 10x RNaseIII buffer, 8 ul sample, 1 ul RNaseIII) 10 min at 37°C. Fragmented samples were precipitated by ethanol and eluted into 4 ul. Adaptor was hybridized and ligated to each sample (10 ul 2x ligase buffer, 2 ul Adaptor mix, 3 ul hybridization solution, 2 ul ligation enzyme) by incubating O.N. at 16°C. Adaptor-ligated RNA was subjected to reverse transcription (4 ul 10x RT buffer, 2 ul dNTP mix, 2 ul RT primer, 1 ul ArrayScript RT, 13 ul water) for 40 min at 42°C. Resulting cDNA was purified using PCR Purification kit (Qiagen) and eluted into 10 ul. cDNA was run on 6% TBE-Urea gel and fragments 150-250 nt in length were excised. Isolated cDNA was amplified for 15 cycles using AmpliTaq DNA polymerase and purified by PCR Purification kit (elution twice in 10 ul). Second round of size selection on PAGE gel was performed and PCR products 110-130 nt in length were excised from gel and isolated.  Purified amplicons were sequenced using Abi SOLiD 4 platform.
attributes.tag: GEO Accession
attributes.value: GSM1142038