RNA-Seq of mus musculus
total RNA was extractedtotal RNA was extracted from tissue samples. Nanodrop2000 was used to detect the concentration and purity of the extracted RNA. Agarose gel electrophoresis was used to detect RNA integrity, and Agilent5300 was used to determine RQN value. A single library construction requires a total RNA of 1 g, a concentration of  30 ng/L, RQN > 6.5, and OD260/280 between 1.8 and 2.2. Oligo dT enriched mRNAThe 3 'end of eukaryotic mRNA has A ployA tail structure, and the A-T base pairing of the ployA with magnetic beads with Oligo(dT) can be separated from the total RNA for analysis of transcriptome information. Fragmented mRNAThe second-generation high-throughput sequencing platform is designed to sequence short sequence fragments, and the enriched mRNA is a complete RNA sequence with an average length of several kb, so it needs to be interrupted randomly. By adding fragmentation buffer and choosing appropriate conditions, mRNA could be randomly broken into small fragments of about 300bp. Reverse synthesis of cDNAUnder the action of reverse transcriptase, a single strand cDNA was synthesized from mRNA template by random primers, and then a stable double-strand structure was formed by double-strand synthesis. Connect the adapterThe double-stranded cDNA structure was A sticky End, which was supplemented into a flat end by adding End Repair Mix, and then an A base was added to the 3 'end to facilitate the addition of adapter sequence. Fragment screening and library enrichmentAfter the adapter connection, the product was purified and the fragment sorted. The sorted product was amplified by PCR and the final library was purified.