GSM1854080: Holstein #4, hour 0; Bos taurus; RNA-Seq
mRNA was collected following challenge with the 5 Prime RNA PerfectPure Extraction Kit. Cell lyaste from duplicate wells were pooled prior to RNA extraction. Massively Parallel sequencing library preparation was performed in accordance to the Illumina TruSeq RNA Sample Prep LT version 2 (RS-122-2001/2002). Breifly, each sample was Poly-A enriched for mRNA followed by a reverse transcription step with SuperScript II (Invitrogen) to convert mRNA to cDNA, then fragmented, end repaired, and adenylated. Illumina indexed adaptors, which contain a universal PCR primer and flow cell binding site, were ligated onto each sample. Each index sequence was unique for the purpose of labelling each sample with its own sequence for individualization and demultiplexing purposes. An Illumina specific PCR master mix was used to perform PCR enrichment/amplification for a predetermined number of cycles using a standard PCR cycle titration in order to keep the number of cycles to a minimum and reduce potential for duplicate sequences in the final data. Generally, 6-10 cycles were required. The enriched PCR product is cleaned using AMPure Beads (Beckman Coulter) and the resulting product is quantified using a Qubit Spectrofluorometer and Aligent Bioanalyzer 2100 High sensitivity DNA chip.
GEO Accession
GSM1854080