Total RNA was harvested from frozen livers using a Qiagen RNAesy kit. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001). The concentration and integrity of the total RNA was estimated by Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, California), and Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA), respectively. Five hundred ng of total RNA was required for proceeding to downstream RNA-seq applications. First, ribosomal RNA (rRNA) was removed using Ribo-Zero™ Gold (Yeast) kit (Epicenter, Madison, WI) using manufacturer's recommended protocol. Immediately after the rRNA removal the RNA was fragmented and primed for the first strand synthesis using the NEBnext First Strand synthesis module (New England BioLabs Inc., Ipswich, MA). The second strand synthesis was then performed using the NEBnext Second Strand synthesis module. Following this the samples were taken into standard library preparation protocol using NEBNext® DNA Library Prep Master Mix Set for Illumina® with slight modifications. Briefly, end-repair was done followed by A-tailing and custom adapter ligation. Post-ligated materials were individually barcoded with unique in-house Genomics Service Laboratory (GSL) primers and amplified through 12 cycles of PCR. Library quantity was assessed by Qubit 2.0 Fluorometer, and the library quality was estimated by utilizing a DNA 1000 chip on an Agilent 2100 Bioanalyzer. Accurate quantification of the final libraries for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5nM and pooled equimolar prior to clustering. Paired-End (PE) sequencing (50 million, 100-bp paired-end reads) was on an Illumina HiSeq2500 sequencer (Illumina, Inc.).
GEO Accession
GSM2365491