Illumina HiSeq 2500 sequencing; ChIP-seq analysis of Esrrb, Nanog, Oct4, and Sox2 in EpiSCs undergoing reprogramming
ChIP-seq analysis of Esrrb, Nanog, Oct4, and Sox2 in EpiSCs undergoing reprogramming
Tet-on Esrrb EpiSCs (GOF18-E3 EpiSCs carrying the Tet-on-Esrrb-IRES-Venus and Pou5f1 [3.5 kb upstream of the TSS]-IRES-Puro transgenes) were maintained in mouse embryonic fibroblast-conditioned media (MEF-CM) containing 20% KnockOut Serum Replacement (KSR) supplemented with 10 ng/ml FGF2 (CM+FGF2) on fetal bovine serum (FBS)-coated dishes. For reprogramming, Tet-on Esrrb EpiSCs were dissociated into single cells using Accutase and plated in CM+FGF2 supplemented with 5 μM Y-27632 on FBS-coated dishes. After overnight culture, the medium was replaced by DMEM/F12 containing 10% KSR supplemented with LIF, 2i (1 μM PD0325901 and 3 μM CHIR99021), and 1 μg/ml Dox. Cells were harvested and cross-linked with 1% formaldehyde in PBS for 10 min at room temperature, and chromatin was sheared using Diagenode Bioruptor (high power, 40 cycles of 30 sec on and 30 sec off) equipped with a water-cooling system (4°C) in sonication buffer (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 0.5% SDS) supplemented with 1× protease inhibitor cocktail (Sigma). Sheared chromatin with an average fragment size of 200‒300 bp was immunoprecipitated in 4× volume of ChIP dilution buffer (10 mM Tris-HCl [pH 8.0], 125 mM NaCl, 0.125% sodium deoxycholate, 1.25% Triton X-100) using Dynabeads protein A or G coupled with the following primary antibodies: mouse anti-Esrrb (PP-H6705-00, R&D Systems); rabbit anti-Nanog (A300-397A, Bethyl Laboratories); goat anti-Oct4 (sc-8628, Santa Cruz); goat anti-Sox2 (GT15098, Neuromics). Beads were washed once with low-salt buffer (20 mM, Tris-HCl [pH 8.0], 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), twice with high-salt buffer (20 mM, Tris-HCl [pH 8.0], 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), twice with RIPA buffer (50 mM, Hepes-KOH [pH 7.6], 250 mM LiCl, 1 mM EDTA, 1% Igepal CA630, 0.7% sodium deoxycholate), and once with TE containing 50 mM NaCl. After incubation with elution buffer (10 mM, Tris-HCl [pH 8.0], 300 mM NaCl, 5 mM EDTA, 0.5% SDS), immunoprecipitated and input DNA were reverse cross-linked and purified. Sequencing libraries were prepared according to the TruSeq ChIP Sample Preparation protocol (Illumina) with size selection of 250‒450 bp.
Experimental Factor: K10+2i/LIF
growth condition
Experimental Factor: doxycycline
stimulus
Experimental Factor: 24
time
Experimental Factor: Sox2
immunoprecipitate