RNA-seq of Mesocricetus auratus
Using ion disruption to break RNA into fragments of approximately 300 bp in length. Choose a segment with a length of 300bp because the length of the connector is fixed. If the length of the interrupted segment is short, it will result in a higher proportion of connector sequences, thereby reducing the proportion of valid data; If the length of the interrupted fragment is long, it is not conducive to the generation of clusters during the machine sequencing process. Using RNA as a template, the first strand of cDNA was synthesized using 6-base random primers and reverse transcriptase, and the second strand of cDNA was synthesized using the first strand of cDNA as a template. After the construction of the library is completed, PCR amplification is used to enrich the library fragments, and then library selection is carried out based on fragment size, with a library size of 450bp. Next, the library is inspected using Agilent 2100 Bioanalyzer, and the total concentration and effective concentration of the library are detected. Then, based on the effective concentration of the library and the required amount of data, the library containing different Index sequences (each sample is added with a different Index, and the off machine data of each sample is distinguished based on the Index) is mixed proportionally. The mixed library was uniformly diluted to 2nM and subjected to alkaline denaturation to form a single stranded library. After RNA extraction, purification, and library construction, the samples were subjected to paired end (PE) sequencing using next-generation sequencing (NGS) technology and Illumina HiSeq sequencing platform.