RNA-Seq for mus musculus: colon tissue from adult male with 3%DSS treatment and aloe-emodin

The mRNA was purified from 1 g total RNA using oligo (dT) magnetic beads followed by fragmentation carried out using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAs were synthesized with random hexamer primers and Reverse Transcriptase (RNase H) using mRNA fragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I, RNAseH, buffer, and dNTPs. The synthesized double stranded cDNA fragments were then adapter-ligated for preparation of the paired-end library. Adaptor-ligated cDNA were used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed on an Agilent Bioanalyzer 4150 system.

本实验从1 g总RNA中利用寡聚(dT)磁珠（oligo (dT) magnetic beads）纯化mRNA，随后在ABclonal第一链合成反应缓冲液中，通过二价阳离子在高温条件下对mRNA进行片段化处理。以mRNA片段为模板，采用随机六聚体引物与反转录酶（Reverse Transcriptase，RNase H）合成第一链cDNA；接着利用DNA聚合酶I（DNA polymerase I）、RNA酶H、缓冲液及dNTPs完成第二链cDNA合成。将合成的双链cDNA片段进行接头连接以制备配对末端测序文库，随后以连接有接头的cDNA为模板进行PCR扩增。扩增产物通过AMPure XP系统纯化，并采用Agilent Bioanalyzer 4150系统评估文库质量。