RNA-Seq of small intestines of anas platyrhynchos
1. RNA quantification and qualification; 2. Library preparation for transcriptome sequencing: Sequencing libraries were generated using an NEBNext Ultra RNA Library Prep Kit for Illumina. After mRNA purification, fragmentation, first and second strand c synthesis, blunt ends conversion, and adenylation of 3 ends of the DNA fragments, an NEBNext Adaptor with a hairpin loop structure was ligated to samples for hybridization preparation. The library fragments were purified using the AMPure XP system, and 3 L of USER Enzyme was incubated with size-selected, adaptor-ligated cDNA. Polymerase chain reaction (PCR) was then performed using Phusion High-Fidelity DNA Polymerase, universal PCR primers, and an index (X) primer. PCR products were purified (AMPure XP system), and library quality was assessed using the Agilent Bioanalyzer 2100 system. 3. Clustering and sequencing: The clustering of index-coded samples was performed on a cBot Cluster Generation System using a TruSeq PE Cluster Kit v3-cBot-HS (Illumina). After cluster generation, the prepared libraries were sequenced on an Illumina Hiseq platform, and 125 bp/150 bp paired-end reads were generated.
SUB4493972
Cile2