alias: GSM2891110
title: GSM2891110: FGL_T; Gallus gallus; OTHER
library_construction_protocol: Total RNA from right and left gonads (ten individuals of each group) was extracted with the SV Total RNA Isolation System Kit according to manual 048 (Promega). Deviating from the protocol, on-column DNAseI digestion of genomic DNA was elongated from original 15 min to 30 min. A second DNaseI digestion of genomic DNA was carried out in solution. Sequencing was performed on Illumina's Genome Analyzer Iix, subsequent base calling carried out by Illumina's GAPipeline. DNase-treated total RNA was subjected to oligo(dT)-based reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen) and an anchored, 5' biotinylated oligo(dT) primer comprising the recognition site for EcoP15I. 3' cDNA fragments were enriched via binding to streptavidin-coated paramagnetic beads (Dynabeads M-270 Streptavidin, Invitrogen) subsequent to cleavage of 5'-CATG sites in reverse-transcribed cDNAs by NlaIII (NEB). An adaptor comprising a known barcoding sequence and a second recognition site of EcoP15I was ligated to the enriched fragments using T4 DNA Ligase (Fermentas). The adaptor-ligated cDNA fragments were released from the beads via digestion by EcoP15I (NEB), end-repaired by KOD DNA Polymerase (Blunting High Kit, Toyobo), and subsequently ligated to a second barcoding adaptor using the T4 Ligase reagent provided with the Ligation high Ver. 2 Kit (Toyobo). Barcoding sequences of the adaptor-ligated cDNA fragments were extended during subsequent PCR-amplification employing Phusion Hot Start High-Fidelity DNA Polymerase (Fermentas) according to the manufacturer's recommendations.
identifiers.namespace: GEO
identifiers.id: GSM2891110