MU374 PT
Libraries were constructed following the manufacturers protocol with reagents supplied in Illuminas Ribo Zero Gold rRNA Removal Kit (Human/Mouse/Rat) and Illumina  Stranded RNA Prep, Ligation Kit.  Prior to rRNA depletion, the sample concentration was determined by Qubit flourometer (Invitrogen) using the Qubit HS RNA assay kit, and the RNA integrity was checked using the Fragment Analyzer automated electrophoresis system.  Briefly, the rRNA is removed from total RNA by hybridization probes using the Ribo Zero kit. The recovered mRNA is fragmented and double-stranded cDNA is generated. Pre-index anchors are ligated to the ends of cDNA.  A subsequent PCR step is used to selectively amplify the anchor-ligated DNA fragments and add indexes and primer sequences for cluster generation.  The final amplified cDNA constructs were purified by addition of Axyprep Mag PCR Clean-up beads.  The final construct of each purified library was evaluated using the Fragment Analyzer automated electrophoresis system, quantified with the Qubit flourometer using the Qubit HS dsDNA assay kit, and diluted according to Illuminas standard sequencing protocol for sequencing on the NovaSeq 6000.