GSM8164164: MGC-803,LV-WT-2; Homo sapiens; RNA-Seq
The samples were delivered to the company (Majorbio Bio-pharm Technology) to prepare RNA samples and conduct high-throughput RNA-seq. Total RNA was extracted from cells using TRIzol Reagent according to the manufacturer's instructions (Invitrogen) and genomic DNA was removed using DNase I (Takara). RNA quality was determined by 2100 Bioanalyser (Agilent Technologies) and quantified using ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD260/280 = 1.8~2.2, OD 260/230≥2.0, RIN≥6.5, 28S:18S≥1.0, > 1 μg) were used to construct a sequencing library. RNA integrity was also confirmed with agarose gel electrophoresis. 1 mg RNA samples were used to construct a sequencing library. Briefly, messenger RNA (mRNA) was first isolated from total RNA samples and then fragmented in a fragmentation buffer. Second, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA, USA) with random hexamer primers (Illumina, CA, USA). Third, the synthesized cDNA was subjected to end-repair, phosphorylation and 'A' base addition according to Illumina's library construction protocol. Then, 200-300 bp cDNA target fragments were isolated using 2% agarose followed by PCR amplification. The isolated cDNA fragments were selected to construct a sequencing library. Libraries were quantified by TBS380 and sequenced with the Illumina HiSeqxten/NovaSeq 6000 sequencer (2×150bp read length) (Illumina).