GSM5739013: R70M1_CGATGT_L008; Homo sapiens; RNA-Seq
Tumors were mechanically disassociated with a homogenizer (Omni International) and then passed through a QIAshredder (Qiagen), gDNA eliminator columns (Qiagen), and RNeasy Plus Mini Kit, as per the manufacturer's instructions, for RNA extraction. Library preparation and sequencing were performed by Novogene. Quality and quantity of RNA were analyzed using NanoDrop, agarose gel electrophoresis, and Agilent 2100. mRNA was purified fromtotal RNA using poly-T oligo-attached magnetic beads. mRNA was first fragmented randomly by fragmentation buffer, followed by NEB library preparation (New England Biolabs). First-strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H−), with second-strand cDNA synthesized using DNA polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads (Beckman Coulter). Exonuclease was used to eliminate overhangs on the double-stranded cDNA. 3′ ends were adenylated, and NEBNext adaptors were ligated in preparation for hybridization. Library fragments were purified with AMPure XP system (Beckman Coulter). Sequencingwas performed byNovogene on an Illumina platform paired-end 150 bp with 20 million reads per sample.