Behnam Abasht - Gene Expression Analysis Using 3’-RNA Sequencing скачать в хорошем качестве

Behnam Abasht - Gene Expression Analysis Using 3’-RNA Sequencing

Watch this webinar on LabRoots at: http://www.labroots.com/webinar/gene-... RNA sequencing (RNA-seq) has revolutionized the study of gene expression in animals, plants and microorganisms. However, because of its high cost, this technology has been mainly used in experiments with limited number of samples. To examine a cost-effective alternative, we used a method, which confines sequencing to the 3’-end of mRNA and produces just one fragment per transcript, resulting in a dramatic decrease in sequencing cost. Total RNA isolated from chicken adipose tissue samples was used for cDNA library preparation using QuantSeq 3’mRNA-seq library Prep Kit. Sixty-one uniquely indexed cDNA libraries were pooled and sequenced on one lane on the Illumina Hiseq 2500. On average, 2.24 million reads per sample were generated, 90.1% of which were mapped to the chicken reference genome (Ensembl Galgal4). For more than 70% of the genes with detectable expression, we redefined the 3’-end and identified alternative polyadenylation sites within the 3’-untranslated regions. To compare gene expression measures between 3’-RNA-seq and RNA-seq technologies, we used data from a subset of 20 samples that were previously used in a RNA-seq study of feed efficiency. The correlation of the log10(fold-change) for gene expression (high- vs. low-feed efficiency birds) between these two methods was 0.90. In conclusion, 3’-RNA-seq is a cost effective method amenable to global gene expression studies at population-level, e.g., expression QTL (eQTL) mapping. Also, it allows for accurate detection of the 3’-end of transcripts, enabling verification of the current gene model annotations and global characterization of alternative.