GSM9700200: CTRL 351; Mus musculus; RNA-Seq
Tumor RNA was extracted via Trizol (ThermoFisher Scientific, Catalog #: 15596026) and transferred into 1.5 mL centrifuge tubes. 100 μL of BCP (Sigma, Catalog #: MKCL6404) was added into tubes for 1 mL of Trizol with vigorous shaking for 15 seconds, and then the tubes were stood for 2-5 minutes at room temperature. Centrifuged at 12,000g, 4 °C for 15 mins and carefully collected the supernatant into a new 1.5 mL centrifuge tube. Added an equal volume of isopropanol. The mixture was reversed and placed at -20 °C for 30 minutes. Centrifuged at 12,000rpm for 15 min at 4°C, the supernatant was discarded carefully, and 1 ml of 75% ethanol was added, prepared with DEPC water, for washing. Centrifuged at 12,000 rpm for 10 min at 4°C and discarded the supernatant. The tubes were stood for 2- 3 minutes at room temperature for drying, then an appropriate amount of RNase-free water (DEPC water) was added until it was completely dissolved. Total RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, USA, Catalog #: E7530L) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer(5X). First-strand cDNA was synthesized using a random hexamer primer and M-MuLV Reverse Transcriptase (RNase H). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3' ends of DNA fragments, NEB Next Adaptor with hairpin loop structure was ligated to prepare for hybridization. To select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with the AMPure XP system (Beverly, USA). Then, 3 µL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, PCR products were purified (AMPure XP system), and library quality was assessed using the Agilent 5400 system (Agilent, USA) and quantified by QPCR (1.5 nM). The qualified libraries were pooled and sequenced on Illumina platforms with PE150 strategy, according to effective library concentration and data amount required.