GSM8493735: SW780-DMSO; Homo sapiens; RNA-Seq
Agarose gel electrophoresis: analyze sample RNA integrity and whether there is DNA contamination; NanoPhotometer spectrophotometer: detect RNA purity (OD260/280 and OD260/230 ratio); Agilent 2100 bioanalyzer: accurately detect RNA integrity. The starting RNA for library construction is total RNA, with a total amount >=800ng. The library construction kit used in library construction is BGI's NEBNext® UltraTM RNA Library Prep Kit. mRNA with polyA tail is enriched by Oligo(dT) magnetic beads, and then the obtained mRNA is randomly broken in NEB Fragmentation Buffer with divalent cations. Using the fragmented mRNA as a template, random oligonucleotides as primers, cDNA first strand is synthesized in M-MuLV reverse transcriptase system, then RNA strand is degraded by RNaseH and cDNA second strand is synthesized in DNA polymerase I system with dNTPs as raw materials. The purified double-stranded cDNA is end-repaired, A-tailed and ligated with sequencing adapters, and AMPure XP beads are used to select cDNA of about 200bp, PCR amplification and AMPure XP beads purification of PCR products are performed to obtain the library. After the library construction is completed, Qubit2.0 Fluorometer is used for preliminary quantification, dilute the library to 1.5ng/ul, then use Agilent 2100 bioanalyzer to detect the insert size of the library, after the insert size meets the expectation, qRT-PCR is used to accurately quantify the effective concentration of the library (library effective concentration higher than 2nM), to ensure the library quality.