cDNA Library Preparation RNA Extraction and Sequencing

The library construction and sequencing was conducted at Novogene, Inc. (Chula Vista, CA, USA). Briefly, mRNA was selected with poly-T oligo-attached magnetic beads from 3 μg of total RNA per biological replicate, and then used for library preparation with the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). Index codes were added in order to assign sequences to each replicate. Briefly, the mRNA was fragmented and then strand cDNA synthesis was primed with random hexamers. After second strand cDNA synthesis, the transcripts were poly-A tailed for ligation of sequencing adaptors. The library fragments were size-selected for cDNA fragments of 150∼200 bp in length, and the library was amplified by PCR. The index-coded samples were sequenced on an Illumina Hiseq platform to generate 125/150 bp paired-end reads. First, raw data (raw reads) of fastq format were processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads from raw data that were low quality as well as those containing adapter or poly-N. The GC content plus the Q20 and Q30 values were calculated. All analyses were conducted with clean data reads. The galGal4 chicken genome assembly was used as the reference genome for alignments with paired-end clean reads using TopHat v2.0.12 (ftp://ftp.ensembl.org/pub/release81/fasta/gallus_gallus/dna/Gallus_gallus.Galgal4.dna.toplevel.fa.gz). The data are accessible through NCBI GEO accession number {"type":"entrez-geo","attrs":{"text":"GSE131652","term_id":"131652"}}GSE131652.