RNA-seq of mus musculus
Large insert non-strand specific RNA Sequencing included plating, poly-A selection and cDNA synthesis, library preparation (400bp insert), sequencing (101bp paired reads) and a sample identification QC check. Non-strand specific RNA sequencing performed at the Broad Institute uses a large-scale, automated variant of the Illumina Tru Seq RNA Sample Preparation protocol (Illumina: TruSeq Protocol Info). An amount of 200 ng of total RNA is used for starting material. This method uses oligo dT beads to select mRNA from the total RNA sample. The selected RNA is then heat fragmented and randomly primed before cDNA synthesis from the RNA template. The resultant cDNA then goes through Illumina library prep (end repair, base 'A' addition, adapter ligation, and enrichment) using Broad designed indexed adapters for multiplexing. After enrichment, the samples are qPCR quantified and equimolar pooled before proceeding to Illumina sequencing with is done on the Illumina HiSeq 2000 or HiSeq 2500, with sequence coverage to 100M paired reads. All samples are electronically tracked through the process in real-time including reagent lot numbers, specific automation used, time stamps for each process step, and automatic registration. The entire process occurs in a 96-well format, and all pipetting is done by either Bravo or Mini Janus liquid handlers.