GSM2125416: PRE - N6W - 2907; Ovis aries; RNA-Seq
An approximate of 15 mm3 of cryosections was pooled and processed using the RNeasy kit (Qiagen). The quantity and quality of total RNA were evaluated using Qubit® RNA BR Assay Kit (LifeTechnologies, USA) and Agilent RNA 6000 Nano kit with the Agilent 2100 Bioanalyzer instrument (Agilent Technologies Inc., Santa Clara, USA), respectively. RNA sample was accepted for the study when the RIN was ≥7.0. The quantity and quality of total RNA were evaluated using Qubit® RNA BR Assay Kit (LifeTechnologies, USA) and Agilent RNA 6000 Nano kit with the Agilent 2100 Bioanalyzer instrument (Agilent Technologies Inc., Santa Clara, USA), respectively. RNA sample was accepted for the study when the RIN was ≥7.0. The Illumina TruSeq® Stranded total RNA protocol with the TruSeq Stranded total RNA Sample Preparation Kit was used to produce library constructs. The library product was generated from 1 µg total RNA subsequent to the removal of ribosomal RNA with human specific probes with the Epicentre Ribo-Zero™ Magnetic Gold Kit and subsequent fragmentation of the enriched RNA. Then first strand cDNA synthesis was carried out using reverse transcriptase and random hexamers, followed by second strand cDNA synthesis with DNA polymerase. During this step the RNA template was removed and double-stranded cDNA was synthetized in the presence of dUTP in place of dTTP. In a next step the 3’ ends of the blunt fragments were adenylated and multiple indexing adapters were ligated to the ends of double strand cDNA. Finally, the cDNA was amplified using PCR in order to both enrich DNA fragments having adapter molecules on both ends and amplify the amount of DNA in the library. Quantification and a quality of generated libraries were determined by qPCR with Illumina adapter specific primers using Roche LightCycler® system (Roche Diagnostics, Basel, Switzerland) and on an Agilent Technologies 2100 Bioanalyzer with DNA-specific chip, respectively. Diluted libraries (10 nM) were pooled and were further used for cluster generation according to the manufacturer’s recommendations using TruSeq SR Cluster Kit v3-cBot-HS reagents and sequenced with TruSeq SBS Kit v3-HS reagents on Illumina HiSeq 2500 in the high output mode. On average 50’000’000 reads were sequenced per library.
GEO Accession
GSM2125416