GSM7611278
RNA was extracted from thawed tissue using the Qiagen RNAeasy Microarray Tissue kit with RWT buffer. 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 and tissue was added. The TissueLyzer was operated for 3 min at 50Hz, allowed to come to a stop, and repeated. Tubes were spun down before opening. Lysate was transferred to a clean 2 ml tube. RNA was isolated through phase separation. 200 µl pf chloroform was added, following by vigorous shaking. Tubes were incubated at RT for 3 min and centrifuged at 12,000 g (max speed) at 4 °C for 15 min. The aqueous phase was transferred to a new tube and total RNA (binding and washing with the RNAeasy column) was purified. 600 µl (1 volume) of 100% EtOH was added. 600 µl was transferred to an RNAeasy mini spin column placed inside a 2ml collection tube and centrifuged 15 s at max speed at RT. This was repeated with the remaining 600 µl. 500 µl of Buffer RWT (bought separately from Qiagen) was added. The tube was centrifuged for 15 s at max speed at RT. The flow through was discarded and the collection tube was kept. 500 µl of buffer RPE was added. The tube was centrifuged for 15 s at max speed at RT. The flow through was discarded and the collection tube was kept. 500 µl of buffer RPE was added. The tube was centrifuged for 2 min at max speed at RT. The flow through was discarded and the collection tube was kept. The spin column was placed on a new collection tube and centrifuged at max speed for a minute to prevent EtOH carryover. RNA was then eluted, and the column was placed into a 1.5 ml Eppendorf tube. 30 µl of RNAse free water was added and the tube was centrifuged for 1 min at max speed at RT. The volume in the tube was pipetted back into the column and the previous step was repeated. 1 µl was used for quantification with the Nanodrop Spectrophotometer. Following quantification, RNA was frozen at -80 °C. The integrity of total RNA was assessed using Fragment Analyzer (Advanced Analytical Technologies, Inc, Ankeny, IA) with the High Sensitivity RNA Analysis Kit (Advanced Analytical Technologies, Inc). 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. All RNA samples had RQNs ranging from 8.8 to 10 and 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 mg 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 (Advanced Analytical Technologies, Inc). The concentrations of RNA libraries were measured by StepOnePlus Real-Time PCR System (ThermoFisher Scientific, San Jose, CA) with the KAPA Library Quantification Kit (Kapa Biosystems, Wilmington, MA). The libraries were diluted to 2 nM with Tris buffer (10 mM Tris-HCl, pH 8.5) and denatured with 0.1 M 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 the HiSeq SBS kit v4 (Illumina). DNA was paired-end sequenced with a read length of 100 bp and an average sequencing depth of 52 million reads per sample.