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.