RNA-seq of capra hircus:perirenal fat of 10 days
First,RNA samples need to be detected. After the RNA samples are qualified, the eukaryotic mRNA is enriched with magnetic beads with Oligo(dT).Following fragmentation buffer is added, mRNA is broken into short fragments. mRNA is used as template and a cDNA is synthesized by random hexamers with six base.Then, buffer solution, dNTPs, DNA Polymerase I and RNase H were added to synthesise two-strand cDNA. Then, AMPure XP beads were used to purify double-strand cDNA.Purified double-stranded cDNA ends are repaired, A-tails are added and sequencing adapters are connected. Then AMPure XP beads are used to select segment sizes.Finally, PCR amplification was performed, and AMPure XP beads were used to purify the PCR products to get the final library.After the completion of library construction, the library was initially quantified using Qubit 2.0 and diluted, followed by Agilent 2100 to detect the size of inserted fragments in the library. After the inserted fragments met the expectation, the effective concentration of the library was accurately quantified using Q-PCR method to ensure the quality of the library.After library detection is qualified, different library pooling will be conducted to flowcell according to the effective concentration and the demand of target offline data volume. After clustering cBOT, sequencing will be performed using Illumina high-throughput sequencing platform Novaseq 6043.
D10-1_pBAT-2