2.2 Sample collection and sequencing of Hi-C experiment
According to previous studies (Barutcu et al., 2015; Battulin et al., 2015; Jiang et al., 2020), we sampled chicken livers from one Wenchang chicken (a laying hen of 43 weeks form Wenchang, Hainan), and one Lindian chicken (a laying hen of 42 weeks form Lindian, Heilongjiang) from their national natural poultry genetic resources conservation farm with tropical and frigid temperature zone, respectively. Then Chicken livers were fixed with 1% formaldehyde solution in MS buffer (10 mM potassium phosphate, pH 7.0; 50 mM NaCl; 0.1M sucrose) at room temperature for 30 min in a vacuum. After fixation, the livers were incubated at room temperature for 5 min under vacuum in MC buffer with 0.15 M glycine. Approximately 2 g fixed tissue was homogenized with liquid nitrogen and resuspended in nuclei isolation buffer and filtered with a 40-nm cell strainer to obtain liver cells. Then the procedures for enriching nuclei from flow-through and subsequent denaturation were done according to a 3C protocol established for maize.
Chromatin was digested for 16 h with 400 U HindIII restriction enzyme at 37 °C. DNA ends were labeled with biotin and incubated at 37 °C for 45 min, and the enzyme was inactivated with 20% SDS solution. DNA ligation was performed by the addition of T4 DNA ligase and incubation at 16°C for 4~6 h. After ligation, proteinase K was added to reverse cross-linking during incubation at 65 °C overnight. DNA fragments were purified and dissolved in 86μL of water. Unligated ends were then removed. Purified DNA was fragmented to a size of 300-500 bp, and DNA ends were then repaired. DNA fragments labeled by biotin were finally separated on Dynabeads® M-280 Streptavidin (Life Technologies). Hi-C libraries were controlled for quality and sequenced on an Illumina Hiseq X Ten sequencer.
2.3 Hi-C reads mapping and filtering and generation of contact matrices
Firstly, low quality paired reads (Reads with ≥ 10% unidentified nucleotides (N); > 10 nt aligned to the adaptor, allowing ≤ 10% mismatches; > 50% bases having phred quality < 5; and putative PCR duplicates generated in the library construction process) were removed, which mainly result from base-calling duplicates and adaptor contamination. Then the high quality paired-end Hi-C reads were mapped to Gallus_gallus-5.0 and filtered using HiCUP v0.5.10 (Steven et al., 2015). HiCUP removed sequences representing experimental Hi-C artifacts and other uninformative di-tags, since even a small number of invalid di-tags could lead to incorrect conclusions being drawn concerning genomic structure.
The genome was divided into 1Mb bins, and the read pair numbers in two regions were counted as the observed interactions by Hicpipe, and the expected interactions were also calculated by this software. The norm interactions were computed by observed interactions divided by expected interactions. We used the norm interactions for every two bins to produce norm contact matrix.
2.4 Identification of compartment A/B
Identification of compartment A/B was performed using the 1Mb interaction matrix as previously described (Wu et al., 2017). The Eigenvector value of two Hi-C samples were calculated by hiclib. Bins with positive values were defined as compartment A, otherwise B.