Arna 6000 nano labchip kit for bioanalyzer 2100

Total RNA was extracted from 40 frozen breast muscle samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. An ARNA 6000 Nano LabChip Kit for Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA) was used to determine the purity and concentration of RNA. Forty cDNA libraries were created by reverse transcription using an mRNA-Seq Sample Preparation Kit (Illumina, San Diego, CA, USA). A HiSeq 2500 instrument was used for paired-end sequencing (Illumina). Raw data (raw reads) in fastq format were filtered to ensure quality. Reads with adapter sequences and low quality were eliminated and all reads containing A bases and reads with N ratios >10 % were eliminated to ensure quality. Using Hisat2 with the default settings, clean paired-end reads were aligned to the chicken reference genome (version: GCF_016699485.2). Stringtie (v2.1.5, http://ccb.jhu.edu/software/stringtie/, accessed on 3 September 2021) was used to assemble transcripts. Gene expression levels were estimated using counts per million (CPM).