title: GSM6463566: P90 wildtype homecage control harvested at ZT hour 5, replicate 4 [WTHC5_P90_PFC_4]; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted from thawed tissue using the Qiagen RNAeasy Microarray Tissue kit with RWT buffer. The tissue was lysed and homogenized using a TissueLyzer. Briefly, 1ml of Qiazol was added to the pre-sterilized 2ml round bottom tubes with one 5mm steel bead inside,before adding the frozen tissue. The tissue was liyed for 2x 2min at 30Hz. Samples were spun down and the clear lysate was transfere to a clean tube. RNA was isolated through phase separation. For this, 200 µl of water saturated chloroform with isoamylalcohol (ChCl3:IAA 24:1) was added followed by vigorous shaking, incubated at rorom temparature of 3 min and then centrifuged at 12,000 g for 15 min at 4C. The aqueous phase was transfered to a new tube and 600ul of 100% EtOH was added. The ethanol-lysate mix was then loaded on a RNAeasy mini spin column and centrifuged at max speed for 30sec at room temparature. The loaded colums were washed using 500 µl of RWT (Qiagen), centrifuged again for 30s at max speed at room temparature. The flowthrough was discarded. The membrane was washed twice with 500 µl of RPE buffer (Qiagen) followed by a 30s (first wash) or 2min (second wash) spin at max speed at room temparature. RNA was eluted from the membrane with 30ul of RNase free water. The RNase free water was loaded directly onto the semi dry membran, centrifuged for one min at max speed at RT. This step was reapeated once by re-loading the eluat and centrigation again for 1min at room temparature. 1ul of the RNA eluat was used to measure concentrations in the Nanodrop. The extraced RNA sampel was aliquoted in 2x15ul aliquots and stored at -80C until further processing. The integrity of total RNA was assessed using Fragment Analyzer (Advanced Analytical Technologies, Ankeny, IA) with the High Sensitivity RNA Analysis Kit.  RNA Quality Numbers (RQNs) from 1 to 10 were assigned to each sample to indicate its integrity or quality.  RNA samples with RQNs ranging from 7 to 10 were used for RNA library preparation with the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA).  Briefly, mRNA was isolated from 2.5 µg of total RNA using poly-T oligo attached to magnetic beads and then subjected to fragmentation, followed by cDNA synthesis, dA-tailing, adaptor ligation and PCR enrichment.  The sizes of RNA libraries were assessed by Fragment Analyzer with the High Sensitivity NGS Fragment Analysis Kit.  The concentrations of RNA libraries were measured by StepOnePlus Real-Time PCR System (ThermoFisher Scientific, San Jose, CA) with the KAPA Library Quantification Kit (Kapabiosystems, Wilmington, MA).  The libraries were diluted to 2 nM with RSB (10 mM Tris-HCl, pH8.5) and denatured with 0.1 N NaOH.  Eighteen pM libraries were clustered in a high-output flow cell using HiSeq Cluster Kit v4 on a cBot (Illumina).  After cluster generation, the flow cell was loaded onto HiSeq 2500 for sequencing using HiSeq SBS kit v4 (Illumina).  DNA was sequenced from both ends (paired-end) with a read length of 100 bp.