GSM5173185: M2_2; Homo sapiens; RNA-Seq
Total RNA was extracted using TRIzol and purified using RNeasy MinElute Cleanup Kit (Qiagen) according to the manufacturer's instructions. Concentration and quality of extracted RNA were measured and quality control (QC) were done following three main methods:  (1) Nanodrop: Preliminary quantitation  (2) Agarose Gel Electrophoresis: tests RNA degradation and potential contamination  (3) Agilent 2100: checks RNA integrity and quantitation Libraries from mRNA were prepared using 1 μg of RNA starting material and NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (Illumina, CA, USA) according to the manufacturer's protocol. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.  The quality control of library consists of three steps:  (1) Qubit 2.0: tests the library concentration preliminarily.  (2) Agilent 2100: tests the insert size.  (3) Q-PCR: quantifies the library effective concentration precisely.