Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: RNA was extracted from thawed tissue using the Qiagen RNAeasy Microarray Tissue kit with RWT buffer. Lyse and homogenize the tissue using a TissueLyzer: Add 1ml of Qiazol to the pre-sterilized 2ml round bottom tubes with one 5mm steel bead inside, then add the tissue. Operate the TissueLyzer for 3 min at 50Hz, let it come to a stop and repeat. Spin down before opening the tubes. Transfer lysate to a clean 2 ml tube. Isolate RNA through phase separation. Add 200 µl pf chloroform and shake vigorously. Incubate at RT of 3 min. Centrifuge at 12,000 g (max of centrifuge) for 15 min at 4 C. Transfer aqueous phase to a new tube. Purify Total RNA (Binding and washing with the RNAeasy column). Add 600 µl (1 volume) of 100% EtOH. Transfer 600 µl to and RNAeasy mini spin column placed inside a 2ml collection tube, centrifuge 15s at max speed at RT. Repeat with remaining 600 µl. Add 500 µl of buffer RWT (buy separately from Qiagen) centrifuge 15s at max speed at RT, discard the flow through, keep the collection tube. Add 500 µl of buffer RPE 15s at max speed at RT, discard the flow through, keep the collection tube. Add 500 µl of buffer RPE 2min at max speed at RT, discard the flow through, discard the collection tube. Place the spin column on a new collection tube and centrifuge at max speed for a minute (to prevent EtOH carryover). Elute the RNA from the column. Place the column into a 1.5 ml Eppendorf. Add 30 µl of RNAse free water, Centrifuge for 1 min at max speed at room temp. Pippette the volume in the Eppendorf back into the column and repeat previous step. Use 1 µl to quantify using Nanodrop Spectrophotometer. After quantification freeze RNA at -80 C. 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.