title: GSM6701006: A2B5_F_Normoxia [A2B5_06]; Rattus norvegicus; RNA-Seq
library_construction_protocol: Total RNA was isolated from each cell type using the Qiagen miRNeasy kit (Cat # 217004) following manufacturer's protocol. In brief, QIAzol Lysis Reagent was used to disrupt and homogenize the cells and the lysate was incubated at room temp for 5 min. Then chloroform was added to the lysate, and shaken vigorously for 15 sec. The mixture was re-incubated for 5 minutes and centrifuged for 15 mins at 14000 RPM at 4 ºC. The upper aqueous phase was collected to a fresh tube and 1.5 volumes of 100% ethanol was added. Then the sample was processed through a RNeasy Mini spin column. On-column DNase digestion was performed to effectively remove DNA during RNA purification by using the RNase-Free DNase set (Qiagen, cat# 79254) according to the manufacturers' instructions. After final column wash, total RNA was eluted out of mini column membranes using 30 µl nuclease free water (Qiagen). Integrity of RNA was confirmed by determining RIN number. Total RNA was delivered to the Center for Genomics at Loma Linda University for library construction and RNA sequencing. The Ovation® Rat RNA-Seq System (NuGEN Technologies, San Carlos, CA) was used per manufacturer's instructions to construct RNA-seq libraries. 100 ng of total RNA was used as input. First and second strands of cDNA were synthesized from total RNA (100 ng) spiked with 1 µl of 1:500 diluted ERCC ExFold RNA Spike-In Mix 2 (Life Technologies, Carlsbad, CA) at a final concentration of 1%. Following primer annealing and cDNA synthesis, end-repair, adaptor index ligation, and strand selection was conducted.  Barcodes with unique indices was used per sample for multiplexing. Ribosomal RNA depletion was performed by using custom InDA-C primer mixture SS5 V8 for rat. Finally, libraries were amplified for 13 cycles (Mastercycler® pro, Eppendorf, Hamburg, Germany), and purified with Agencourt XP beads (Beckman Coulter, Indianapolis, IN).