GSM5049701: T24-NC1; Homo sapiens; RNA-Seq
TRIzol (Thermo Fisher Scientific, Inc.) was used for cells total RNA extraction according to manufacturer's protocol. Then different groups of RNA were used for RNA-seq or qRT-PCR. Strand specific library construction: the method of reverse transcription synthesis of the first strand of cDNA is the same as that of NEB, but the difference is that when the second strand is synthesized, the dTTP in dNTPs is replaced by dUTP, and then the cDNA ends are repaired, A-tail is added, sequencing connector is connected, and length screening is carried out. Then the second strand of cDNA containing u is degraded by user enzyme, and then PCR amplification is carried out to obtain the library. Chain specific library has many advantages, such as obtaining more effective information under the same amount of data; obtaining more accurate gene quantitative, positioning and annotation information; providing the expression level of antisense transcripts and single exon in each isoform. The kit used to build the library was NEBNEXT® Ultra ™ Directional RNA Library Prep Kit for Illumina®。After the construction of the library, qubit2.0 fluorometer was used for preliminary quantification, and the library was diluted to 1.5ng/ul. Then the insert size of the library was detected by Agilent 2100 Bioanalyzer. After the insert size met the expectation, the effective concentration of the library was accurately quantified by QRT PCR (the effective concentration of the library was higher than 2nm) to ensure the quality of the library.