2.8 Transcriptome sequencing and analysis
Liver tissues from 8 chickens (4 LDCs of 42 weeks form Lindian, Heilongjiang and 4 WCCs of 43 weeks form Wenchang, Hainan) were collected from their national natural poultry genetic resources conservation farm and sequenced for transcriptomic analysis. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform and 150 bp paired-end reads were generated. Index of the reference genome (Database: Eensemble_90, version : Gallus_gallus.Gallus_gallus-5.0.dna.toplevel.fa) was built using Bowtie v2.2.3 and the high-quality RNA-seq reads were aligned to the reference genome by the HISAT2 v2.0.4 program with default parameters. HTSeq v0.6.1 was used to count the reads numbers mapped to each gene. And then Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of each gene was calculated based on the length of the gene and reads count numbers (by perl script).