title: GSM2065906: rep2_smit_fastPolII; Saccharomyces cerevisiae; RNA-Seq
library_construction_protocol: Nascent RNA was prepared using a nascent RNA isolation protocol for yeast (Carrillo Oesterreich et al. 2010) and Phenol:Chloroform:IAA, 25:24:1, pH 6.6 extraction. Total RNA was extracted with Phenol:Chloroform:IAA, 25:24:1, pH 6.6 using the RiboPure RNA Purification Kit, yeast (Life technologies). Nascent RNA was 3’ end ligated to the DNA adaptor (/5rApp/NNNNNCTGTAGGCACCATCAAT/3ddC/) and reverse transcribed into cDNA with a primer complementary to the DNA adaptor (ATTGATGGTGCCTACAG). The cDNA solution was diluted by 1:2-1:5 and used as template for gene-specific PCRs. The forward primer was designed to bind to a gene-specific sequence, whereas the reverse primer targets the cDNA 3’ end with the adaptor sequence. Both primers carried Illumina library adaptor overhangs. The individual PCRs were pooled and PCR purified. The pooled cDNA was template for a second PCR, which attached final library adaptors. All PCRs were adjusted in their cycle numbers. Prior to sequencing the double-stranded DNA ranging from ~150 bp (no insert product) to >2 kb was purified by gel electrophoresis. This step was not necessary for size-selected short RNA samples. Final sample purification was done with AMPure beads. Paired-end sequencing of DNA > 200 bp was done after Bioanalyser, Qubit and Kappa library quantification. For 3' end sequencing, 3’ end ligated nascent RNA was heat-fragmented and then reverse transcribed using the DNA adaptor sequence as RT primer. The final paired-end sequencing library was prepared according to in-house protocols of the Yale Center for Genome Analysis. targeted paired-end RNA-Seq
attributes.tag: GEO Accession
attributes.value: GSM2065906