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).