GSM7765994: HepG2 cells FN3K KO, sample 3; Homo sapiens; RNA-Seq
After cell lysis, impurities removal, and inhibition of RNAse activity, total RNA was extracted by using phase separation method from cell debris, and was precipitated by ethanol. Then Agilent bioanalyzer 2100 were used to check RNA integrity and concentration. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. For prokaryotic organisms or eukaryotic organisms' long-non-coding libraries, rRNA is removed using the Ribo-Zero kit that leaves the mRNA. 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.