BS-Pre-EA4(M+EA_mice)
In this project, polyA-bearing mRNA was enriched by magnetic beads, and RNA was broken into 200-300bp fragments by ion breaking. With RNA as template, the first strand of cDNA was synthesized with 6-base random primers and reverse transcriptase, and the second strand of cDNA was synthesized with the first strand of cDNA as template. When the second strand of cDNA was synthesized, the base T was replaced by U, thus achieving the purpose of chain-specific library. Our RNA-Seq project uses chain-specific kit to construct library. Chain-specific library can determine the transcription direction of sense strand and antisense strand, which makes the subsequent analysis results of gene function annotation and gene expression more accurate. After the construction of the library, PCR amplification was used to enrich the library fragments, and then the library was selected according to the fragment size, which was 300-400bp. Then, the library was inspected by Agilent 2100 Bioanalyzer, and then the total concentration and effective concentration of the library were detected. Then, according to the effective concentration of the library and the amount of data required by the library, the libraries containing different Index sequences (adding different Indexes to each sample, and finally distinguishing the off-machine data of each sample according to the index) are mixed proportionally. The mixed library was uniformly diluted to 10nM and denatured by alkali to form a single-stranded library. After RNA extraction, purification, and library building, the libraries were Paired-End (PE) sequenced by Next-Generation Sequencing (NGS) based on Illumina HiSeq sequencing platform.
R7j1061