GSM6449892: Wildtype harvested following 5 hours of sleep deprivation, replicate 2 [RNA-Seq]; Mus musculus; RNA-Seq
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. 1ml of Qiazol was added to the pre-sterilized 2ml round bottom tubes with one 5mm steel bead inside, before adding the tissue. The TissueLyzer was operated for 2x3 min at 50Hz. Samples were spun down prior to opening the tubes. Lysate was transferred to a clean 2 ml tube. RNA was isolated through phase separation. 200 µl pf chloroform was added followed by vigorous shaking. Samples were then incubated at room temperature for 3 min. Next, samples were centrifuged at 12,000 g (max centrifuge) for 15 min at 4 C. The aqueous phase was transferred to a new tube. Total RNA was purified (Binding and washing with the RNAeasy column). 600 µl (1 volume) of 100% EtOH was added. 600 µl was then transferred to an RNAeasy mini spin column placed inside of a 2ml collection tube and centrifuged at max speed for 15s at room temperature. This was repeated with the remaining 600 µl. The loaded columns were washed using 500 µl of RWT (Qiagen) and centrifuged at max speed for 15 s at room temperature. The flow through was discarded and the collection tube was kept. The membrane was washed twice with 500 µl of RPE buffer (Qiagen) followed by 15s (first wash) and 2 min (second wash) centrifugation at max speed at room temperature. The spin column was placed on a new collection tube and centrifuged at max speed for a minute (to prevent EtOH carryover). RNA was eluted from the column. The column was placed into a 1.5 ml Eppendorf. 30 µl of RNAse free water was added and centrifuged at max speed for 1 min at room temperature. The volume was pipetted in the Eppendorf tube back into the column and the previous step was repeated. 1 µl was used to quantify using a Nanodrop Spectrophotometer. The RNA was freezed at -80 C after quantification. 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.  “10” stands for a perfect RNA sample without any degradation, whereas “1” marks a completely degraded sample.  RNA samples with RQNs ranging from 8 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.