Fd0014

The exon 6 and 3 ${}^{\prime }$ flanking region of POU1F1 gene was amplified
using F: 5 ${}^{\prime }$ -CCATCATCTCCCTTCTT-3 ${}^{\prime }$ and R: 5 ${}^{\prime }$ -AATGTACAATGTCCTTCTGAG-3) ${}^{\prime }$
primers (Lan et al., 2007b). The 25  $\mathrm{\mu }\mathrm{L}$ PCR volume contained
100 ng genomic DNA, 0.5  $\mathrm{\mu }\mathrm{M}$ of each primers, $\mathrm{1}\times$  PCR
Buffer, 200  $\mathrm{\mu }\mathrm{M}$ dNTP, 2 mM ${\mathrm{MgCl}}_{\mathrm{2}}$ and 1 U of Taq DNA
polymerase (i-StarTaq^™ DNA Polymerase, iNtRon Biotechnology). The
cycling protocol was 5 min at 95  ${}^{\circ }$ C, 35 cycles of 94  ${}^{\circ }$ C
for 30 s, 54  ${}^{\circ }$ C annealing for 30 s, 72  ${}^{\circ }$ C for 45 s with
a final extension at 72  ${}^{\circ }$ C for 10 min.
PCR products of the POU1F1 gene were digested with 10 U of
AluI and PstI restriction enzymes (FD0014 and FD0614,
Thermo Fisher Scientific) at 37  ${}^{\circ }$ C for 3 h. PCR products and
restriction fragments were electrophoresed on a 2.5 % agarose gel stained
with SafeView^™ Classic (Applied Biological
Materials Inc., Canada).
POU1F1 gene fragments which gave different genotypes were also
sequenced. The sequences of POU1F1 fragments were analyzed by using
the MEGA6 software (Molecular Evolutionary Genetics Analysis, version 6.0;
Tamura et al., 2013) for generating sequence alignments.

Cross‐linking of cells was performed by incubating 1 × 10⁷ cells in phosphate buffer solution (PBS) containing 1% formaldehyde; glycine (0.125 M) was added to stop the reaction. Complexes were precipitated by ethanol at −20°C. Precipitates were dissolved in Tris–EDTA buffer to a total volume of 80 μl and digested by Alui (FD0014, Thermo Fisher, MA, USA) at 37°C for 1 h. Alui was inactivated at 65°C for 20 min and then samples were incubated with T4 DNA ligase (Promega) at 16°C for 5 h. Protein–DNA complexes were de‐cross‐linked by adding SDS and NaCl up to 1% and 0.3 M, respectively, at 55°C overnight. Samples were then treated with proteinase K at 65°C for 1 h. DNA was purified by a kit from TIANGEN Biotech (Beijing, China). To visualise the enhancer–promoter interaction, the purified DNA was PCR‐amplified. Each of the purified PCR products was sequenced to confirm the identity. Primers are listed in Table S1.