title: GSM1254205: hESC_H1_Illumina; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was prepared by TRIzol extraction (Ambion; http://www.ncbi.nlm.nih.gov/pubmed/2440339) and treated with RNase-free DNase I to degrade contaminating gDNA. This was followed by a second extraction with acid-phenol-chloroform and the RNA was precipitated in 0.5M ammonium acetate and 2.5 volumes of ethanol. The integrity of the RNA was tested by a Bioanalyzer RNA Nano assay (Agilent). PolyA RNA was purified from the total RNA (>100 µg) using magnetic oligo-dT beads (Dynal) according to the manufacturer’s recommended protocol. Two different manufacturer’s kits and accompanying protocols for generating full-length cDNAs were employed; Clontech SMARTer PCR and Invitrogen Superscript Full-Length cDNA kit. Each utilizes a retroviral reverse transcriptase to initiate cDNA synthesis at the 3’ polyA tail, but the two kits use different mechanisms to capture the 5’ end information. The Clontech method relies upon template-switching to an exogenous tag oligonucleotide when the reverse transcriptase reaches the 5’ end of the molecule. The Invitrogen method employs an RNase I digestion and subsequent 7-methyl-guanosine affinity step to purify the RNA-DNA hybrids that represent a full copy of the transcript. This is followed by an RNA ligase step to add a defined sequence to the 5’ ends of the polyA RNAs, prior to reverse transcription. For each method, 1 µg of polyA RNA served as the input material into the recommended protocol and the manufacturer methods were followed up through second strand cDNA synthesis. At this point, PCR (12-15 cycles; Phusion DNA polymerase. New England Biolabs) was used to amplify the double-stranded cDNA by using specific primers directed to the defined sequences present at the 5’ and 3’ ends of the cDNAs. Long extension times of 3.5 minutes ensure that longer molecules can be captured as well. The cDNA libraries were assayed by fluorimetry and Bioanalyzer assays. Both of Clontech and Invitrogen kits are reproducible. For example, housekeeping genes at different sizes (ACTB, 1808bp; GADPH, 1310bp; SARS 2291bp; IARS, NM_002161, 4406bp and NM_013417, 4510bp) are detected by full length long reads from the two preps .
attributes.tag: GEO Accession
attributes.value: GSM1254205