10^7 to 5x10^7 E14Tg2a Esrrb-2a-GFPd-IBIH ESCs were resuspended at 3.3x10^6 cells/ml in pre-warmed GMEMb/FCS/LIF and cross-linked for 10 min at RT with 1% formaldehyde (Sigma Cat # F8775-25ML) in the dark. Cross-linking was stopped by adding 0.125mM glycine (5 min, RT), cells were pelleted (5 min, 1000 rpm, 4°C), washed twice with ice-cold PBS and resuspended in PBS/FCS at 10^7 cells/ml. Keeping the samples refrigerated, Esrrb High (Top 10%), Esrrb Medium (15% of the distribution immediately above negative) and Esrrb Negative cells were purified using a FacsARIA cell sorter (Becton, Dickinson). Since gradual loss of fluorescence was observed in fixed cells after prolonged incubation, multiple batches of freshly fixed cells were sorted for a maximum of 45 minutes, a time when loss of fluorescence was not yet noticeable. After sorting, cell purity was determined using the same instrument. Cells were spun down, transferred to 1.5 ml Eppendorf tubes, washed with cold PBS, counted and resuspended at 5x10^6 cells/ml in swelling buffer (5mM Pipes pH8, 85mM KCl) freshly supplemented with 1X protease inhibitor cocktail (Roche Cat # 04 693 116 001)/0.5% NP-40. After 30 min on ice with occasional shaking, nuclei were centrifuged (600g, 5 min, 4°C), washed in TSE (0.1% SDS, 1% Triton, 2mM EDTA, 20mM Tris-HCl pH8)/150mM NaCl, freshly supplemented with 1X protease inhibitor cocktail, and resuspended in the same buffer at 107 cells/ml. Samples were sonicated in 1.5ml tubes using a Bioruptor (Diagenode) (4 x 10min cycles (each divided into 30s ON-30s OFF subcycles) at maximum power in circulating ice-cold water. Chromatin was then centrifuged (30 min, 14,000 rpm, 4°C) and the supernatant stored (-80°C) until use. 5ul was used to quantify chromatin concentration and check the DNA fragment size on a 1.5% w/v 0.5x TAE agarose gel. The typical fragment size was 100-250bp. For each ChIP experiment a total of 5-10 ug of chromatin was used, pooling material generated from multiple sorting experiments. Chromatin was thawed and pre-cleared (90 min rotating on-wheel, 4°C) in 1 ml TSE/150mM NaCl containing 50µl of a 50% slurry of pA/pG sepharose beads (Sigma Cat # P9424-5ML, P3296-5ML), previously blocked with 500µg/ml BSA (Roche, Cat # 5931665103) and 1µg/ml of yeast tRNA (Invitrogen, Cat # AM7119). Pre-cleared chromatin was centrifuged (3000rpm, 1 min) in a benchtop microcentrifuge and the supernatant transferred into fresh tubes. 10ul of diluted chromatin was set apart for input DNA extraction and precipitation. Immunoprecipitations with anti-Nanog rabbit polyclonal (made in-house) (0.5 ug/IP) and goat polyclonal anti-Oct4 (Santa Cruz Biotechnology, Cat # SC-8628) (1 ug/IP) antibodies were performed by rotating on-wheel (4°C, overnight) in a final volume of 500µl TSE/150mM NaCl. Immunocomplexes were recovered by rotating (4h, 4°C) with 25ul of a 50% slurry of pA/pG sepharose beads (Sigma Cat # P9424-5ML, P3296-5ML) or 20µl pA/pG magnetic Dynabeads (Life Technologies Cat # 10001D, 10003D) (30 mg/ml), (blocked as above). Beads were recovered by centrifugation or magnetic separation and washed sequentially (5 min rotation, RT) with 1ml of the following buffers: TSE/150mM NaCl (3x), TSE/500mM NaCl (once), TSE/750mM NaCl (once), TSE/1000mM NaCl (once), washing buffer (10mM Tris-HCl pH8, 0.25M LiCl, 0.5% NP40, 0.5% Na-Deoxycholate, 1mM EDTA), and twice in TE (10mM Tris-HCl pH8, 1mM EDTA). After the last wash, a 2-step elution was performed by incubating beads in 150µl 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH8 (15 min; 65°C) after vigorous vortexing. Eluates were collected (1 min, 14,000 rpm), a second elution performed under the same conditions and both eluates pooled. For both immunoprecipitated and input chromatin samples, crosslinking was reversed by incubation (65°C, overnight) followed by proteinase K treatment (5 ul - Invitrogen Cat # AM2546), phenol/chlorophorm extraction and ethanol precipitation using 40ug of glycogen as a carrier. Material from three independent ChIPs was pooled, resuspended in 10ul H2O and used as a template for library preparation using a MicroPlex Library Preparation Kit (Diagenode Cat # C05010010) following the manufacturer instructions. The library preparation and synthesis steps were performed using a benchtop thermocycler (Biorad). For both Oct4 and Nanog ChIP libraries Esrrb High, Esrrb Medium and Esrrb Negative samples were labelled with indexing reagents n. 4, 6 and 12 respectively. Before library amplification, Picogreen (Life Technologies Cat # P11496) was added at working concentration (1:200 from stock) in the amplification mix. 75ul of mix for each sample was transferred to three wells of a 384 well qPCR plate (Roche) and library amplification performed using a 480 LightCycler (Roche). Amplifications were stopped before the reaction reached plateau and DNA concentration quantified. In case the total amount of DNA was below 500ng, 4 additional cycles of amplification were performed. Amplified material was purified using Agencourt AMPure® XP magnetic beads (Beckman Coulter, Cat # No. A63880) and eluted in 30ul TE. Samples were submitted to the GenePool core facilities (University of Edinburgh) for quantification and analysis of fragment size on an Agilent 2100 Bioanalyser. Fragment sizes ranged from 200-450 bp in all samples. Equimolar amounts of Oct4 or Nanog ChIP libraries from Esrrb High, Esrrb Medium and Esrrb Negative sorted cells were pooled and sequenced on a single Illumina HiSeq 2500 flowcell.