title: GSM8010154: T6; Homo sapiens; RNA-Seq
library_construction_protocol: Upon harvest, tumors were stored in Qiagen AllProtect at −80°C. Samples were processed using Qiagen Tissuelyser LT along with Qiagen AllPrep DNA/RNA extraction kit. Samples were quantified and quality control was performed before libraries were prepared. Library preparation and sequencing was performed by Novogene with the Illumina PE150 platform at a read depth of 40 million reads (Novogene Corp., Chula Vista, CA).. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads, followed by random fragmentation via addition of fragmentation buffer. Library construction was performed using the NEBNext® UltraTM RNA Library Prep Kit. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext® Adaptor with hairpin loop structure is ligated to prepare for hybridization. To preferentially select cDNA fragments of 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter, Beverly, USA). The final library was then obtained by PCR amplification and purification of PCR products by AMPure XP beads.