RNA-Seq of 3-month-old female Ctns-/- mouse fed with standard diet: kidney
Total RNA sample was purified by oligo(dT)-attached magnetic beads. mRNA molecules were fragmented into small pieces using fragmentation reagent. First-strand cDNA was generated by First Strand Master Mix and Super Script II through reverse transcription, then add Second Strand Master Mix to synthesize the second-strand cDNA. The purified fragmented cDNA was combined with A-Tailing Mix, and incubated. Then combine the Adenylate 3'Ends DNA, RNA Index Adapter and Ligation Mix, and incubate. Purify the product DNA with Ampure XP Beads. Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix were performed to enrich the cDNA fragments. Then the PCR products were purified with Ampure XP Beads. The libraries were assessed quality and quantity in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The qualified libraries were amplify on cBot to generate the cluster on the flowcell. And the amplified flowcell will be sequenced pair end on the HiSeq System (HiSeq 4000 SBS KIT, Illumina).