GSM3097856: 322P1M; Bos taurus; RNA-Seq
Total RNA was isolated from 50mg of biopsy samples using QIAzol (Qiagen, UK). Tissue samples were homogenised in 1mL of QIAzol reagent using a rotor-strator tissue lyser (Qiagen, UK) and chloroform (Sigma-Aldrich Ireland, Dublin, Ireland). RNA was subsequently precipitated and purified using the RNeasy plus Universal kit (Qiagen, UK) according to the manufacturer's guidelines, which included a step to remove any contaminating genomic DNA. The quantity of the RNA isolated was determined by measuring the absorbance at 260nm using a Nanordrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA). RNA quality was assessed on the Agilent Bioanalyser 2100 using the RNA 6000 Nano Lab Chip kit (Agilent Technologies Ireland Ltd., Dublin, Ireland). RNA quality was also verified by ensuring all RNA samples had an absorbance (A260/280) of between 1.8 to 2.0 and RIN (RNA integrity number) values of between 8 and 10 were deemed high quality. Any samples that had a (A260/280) absorbance of less than 1.8 were cleaned using Zymo Research RNA clean & concentrator kit (Cambridge Biosciences, UK).  High quality RNA samples were selected for cDNA synthesis. cDNA libraries were prepared from high quality RNA following the manufacturer's instructions using the Illumina TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA, USA). For each sample, 1 μg of RNA was used for cDNA library preparation. Briefly, mRNA was purified from total RNA and subsequently fragmented. First strand cDNA was synthesised using Superscript II Reverse Transcriptase (Applied Biosystems Ltd., Life Technologies) followed by second strand synthesis using components of the Illumina TruSeq RNA sample prep kit. Adaptors were ligated to the cDNA which was subsequently enriched by 15 cycles of PCR. Libraries were validated on the Agilent Bioanalyser 2100 using the DNA 1000 Nano Lab Chip kit. cDNA concentration was assessed using Nanordrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA) and samples with >25ng/μl were deemed suitable for further analysis. Following quality control procedures, individual RNAseq libraries were pooled based on their respective sample-specific 6bp adaptors and sequenced at 50bp/sequence single-end read using and Illumina HiSeq 2500 sequencer.
GEO Accession
GSM3097856