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Supporting Information Supporting Information Rugg-Gunn et al. 10.1073/pnas.0914507107 SI Methods Chromatin Immunoprecipitation (ChIP). Different ChIP procedures Embryo Handling and Dissections. All animal work was performed are necessary for different applications/analyses. For example, in accordance with guidelines established by the Canadian where possible we used native ChIP to examine histone mod- Council on Animal Care. Mice used in this study included wild ifications, whereas fixed ChIP is needed to examine Polycomb type (strain ICR) and B5/EGFP, in which enhanced green binding and for sequential ChIP. Similarly, cChIP is used when ~ fluorescent protein is ubiquitously expressed (1). examining 1,000 cells. Importantly, the same assay was always Embryos were collected at appropriate time points from timed used within a figure or experiment, or when comparing cell lines. natural matings. Embryos on E3.5 were collected by flushing In addition, all key findings were replicated using alternative ChIP fi dissected uteri with M2 media (Specialty Media, Millipore). approaches where possible, con rming that the different meth- Embryos on E4.5, E5.5, and E6.5 were dissected from decidua in odologies do not affect the outcome of the experiment. In par- PBS with Ca2+ and Mg2+. Reichert’s membranes were removed ticular, the low prevalence of H3K27me3 sequence reads in TS fi from E5.5 and E6.5 embryos using 30-gauge needles before and XEN cells was con rmed for a subset of developmentally transfer to CO -calibrated DMEM/F12 supplemented with 10% important genes by quantitative PCR analysis of ChIP DNA. We 2 fi fi FBS. Isolation of epiblast (EPI), extraembryonic ectoderm also veri ed that our ndings were not unique to our methodology by obtaining highly similar results using an alternative H3K27me3 (ExE), and visceral endoderm (VE) was carried out as described fi (2). Briefly, E5.5 embryos were staged so that the proamniotic antibody and by performing ChIP on xed chromatin. Native (unfixed) ChIP was performed as described (5). Briefly, cavity was restricted to the embryonic portion of embryo, washed – four times in ice-cold PBS without Ca2+ and Mg2+, followed by 10 50 million cells were lysed in 0.2% (vol/vol) IGEPAL CA-630 (Sigma) for 10 min on ice, nuclei were collected by centrifugation incubation for 15–20 min in Hank’s based cell dissociation buffer at 10,000 × g for 20 min at 4 °C and suspended in digestion buffer (Invitrogen) on ice. Embryos were transferred to ice-cold drops at 1 mg/mL. Chromatin aliquots (0.5 mg in 500 μL) were digested of Ca2+- and Mg2+-free flushing and handling media (Specialty with 10 U micrococcal nuclease (GE Healthcare) for 6–9 min Media, Millipore) where the VE was reflected off the embryo at 37 °C and soluble chromatin recovered by overnight dialysis using 30-gauge needles. The remainder of the embryo was bi- followed by centrifugation. Successful chromatin fractionation sected along the embryonic/extraembryonic axis, and, if distin- (sample contains predominantly mononucleosomes to tetranu- guishable, the EPC was removed from the ExE and discarded. cleosomes) was verified by agarose gel electrophoresis. Frag- E6.5 embryos were processed similarly, except that EPCs were mented chromatin (35 μg per ChIP) was immunoprecipitated retained and all maternal tissue was removed. Isolated EPI, ExE, overnight at 4 °C with 2–10 μg of one of the following antibodies: VE, and EPC were pooled from multiple embryos and litters. – μ H3K4me3 (Abcam, ab8580), H3K9me2 (Millipore, 07 212), For cChIP analysis, tissues were snap-frozen in 50 L PBS sup- H3K9me3 (Abcam, ab8898), 1 μL H3K9me3 (6), H3K27me3 plemented with protease inhibitors (mini complete-EDTA free, (Millipore, 07–449), 1 μL H3K27me3 (7), H3K79me3 (Abcam, − Roche) and 5 mM sodium butyrate (Sigma) and stored at 80 °C ab2621), H4K20me3 (Millipore, 07–463), H3K9 acetylation until use. For mRNA expression analysis, tissues were homoge- (Millipore, 07–352), or rabbit anti-mouse IgG (Jackson, 315–005- μ nized in 800 L TRIzol (Invitrogen) and processed immediately 003). Protein A-sepharose (100 μL of 50% (vol/vol) slurry; GE ’ following manufacturer s instructions for small cell samples. Healthcare) was added to each sample and rotated for 4 h at 4 °C. Unbound material was removed, sepharose beads washed in salt Cell Culture. Mouse ES cell lines, R1 (passages 12–15) and – buffer (increasing sodium chloride concentration from 75 to E14TG2a (passages 19 21), were cultured on gelatin-coated 175 mM) and chromatin eluted from the beads with two 15-min surfaces in standard ES cell media (DMEM supplemented with μ incubations in 1% SDS at room temperature. DNA from bound, 15% FBS, 1 mM sodium pyruvate, 50 U/mL penicillin, 50 g/mL unbound, and input samples was extracted by two rounds of μ streptomycin, 50 M 2-mercaptoethanol, 0.1 mM nonessential phenol/chloroform and precipitated with isopropanol overnight amino acids (NEAA), 2 mM glutamax (all from Invitrogen), and at −20 °C with 30 μg glycogen (Roche) as carrier. Air-dried DNA 1,000 U/mL LIF. pellets were reconstituted in TE buffer. Throughout the pro- – – TS cell lines, A4 (passages 6 15) and G3 (passages 7 14), were cedure, all solutions were ice-cold and freshly supplemented with × tg/tg derived from E3.5 blastocysts obtained from ICR B5/EGFP protease inhibitors (mini complete-EDTA free, Roche) and matings as previously described (3). TS cells were maintained in 5 mM sodium butyrate. RPMI (Sigma) supplemented with 20% FBS, 1 mM sodium Formaldehyde crosslinked and sonicated chromatin was ana- pyruvate, 50 U/mL penicillin, 50 μg/mL streptomycin, 50 μM2- lyzed using the ChIP assay kit (Millipore) following manufacturer’s mercaptoethanol, 2 mM glutamax (all from Invitrogen), 25 ng/ protocol. Chromatin from 1 million cells was immunoprecipitated mL FGF4 (R&D Systems), and 1 μg/mL heparin (Sigma), with using the following antibodies: 3 μg Rnf2 (Abnova, H00006045- 70% of the media preconditioned by mitotically inactivated M01), 3 μg Ezh2 (Active Motif, 39103), 5 μg Ezh2 (Abnova, 5 embryonic fibroblasts. To induce TS cell differentiation, 1 × 10 PAB0648), 5 μg H3K27me3 (Millipore, 07–449), 5 μg Eed (Mil- cells were plated onto a 6-cm plate in standard TS cell conditions lipore, 09–774), and 5 μg anti-mouse IgG (Jackson 315–005-003). for 24 h, rinsed twice with PBS, and cultured for 6 days in un- Sequential ChIP was performed as described (8) with modi- conditioned TS cell media without FGF4 and heparin. Media fication to the first elution. Briefly, formaldehyde crosslinked and was changed every 2 days. sonicated chromatin (50 μg) was immunoprecipitated for 2 h at XEN cell lines, F3 (passages 7–12) and F4 (passages 7–14), were 4 °C using 2.4 μg H3K4me3 (Abcam, ab8580) that was prebound derived from E3.5 blastocysts obtained from ICR × B5/EGFPtg/tg to protein A Dynabeads (Invitrogen). Immune complexes were matings as previously described (4). XEN cells were maintained washed three times with RIPA and once with TE. The first on gelatin-coated surfaces in TS cell media (70% preconditioned) elution was carried out for 30 min at 37 °C with 30 μL20mM without FGF4 or heparin. Protocols can be found at http://www. Tris–HCl, pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, and sickkids.ca/research/rossant/custom/stemCells.asp. 20 mM DTT. Eluted chromatin was diluted 50-fold with RIPA and Rugg-Gunn et al. www.pnas.org/cgi/content/short/0914507107 1of15 immunoprecipitated overnight at 4 °C with 2.4 μg H3K27me3 Immunofluorescence. Embryos were processed as described pre- (Millipore, 07–449), 0.5 μL H3K9me3 (6), or 2.4 μg anti-mouse viously (12). Primary antibodies included: Cdx2 (1:200, Biogenex, IgG (Jackson 315–005-003). After washing as before, the second CDX-88), H3K4me3 (10 μg/mL, Abcam, ab8580), H3K27me3 elution was carried out for 2 h at 68 °C in 250 μL elution buffer (6 μg/mL) (7), and H3K9me3 (1:300) (6). Secondary antibodies (without DTT) supplemented with 50 μg/mL proteinase K. An included: anti-rabbit Alexa546 and anti-mouse Alexa488 (5 μg/mL; additional elution was carried out with a further 250 μL for 5 min Invitrogen). Nuclei were stained with Draq5 (10 μM; Invitrogen) or at 68 °C and DNA was recovered by phenol/chloroform extrac- Hoechst 33342 (1 μg/mL, Invitrogen). Images were collected by tion and ethanol precipitation. confocal microscopy as previously described (12). Quantification of immunoprecipitated DNA was performed by qPCR. For each sample, immunoprecipitated DNA was calcu- Western Blot. Protein extracts (20 μg per lane) were resolved by lated as a percentage of total input DNA and normalized to an SDS/PAGE and transferred on to PVDF membranes. Mem- intergenic region. Primers were designed to target the promoter branes were blocked with 3% milk and incubated with anti-Rnf2 region of each gene. Primer sequences are detailed in Table S1. (1 in 1,000; Abnova, H00006045-M01), anti-Eed (1 in 500; Topreparesamplesfor Illumima sequencing,the concentrationof Millipore, 09–774), anti-Ezh2 (1 in 2,000; Abnova, PAB0648), immunoprecipitated DNA was determined using Quant-iT Pico- anti-Ezh1 (1 in 500; Thermo, PA1-41114), anti-Suz12 [1 in 1,000 Green dsDNA reagent (Invitrogen) and 100 ng of each sample was (diluted in 5% BSA/TBST), Cell Signaling, 3737S], anti- sent to BC Cancer Agency Genome Sciences Centre, Vancouver, H3K4me3 (1 in 2,000, Abcam, ab8580), anti-H3K27me3 (1 in Canada, where the service was performed. Sample processing and 1,000, Millipore, 07–449), anti-H3K9me3 (1 in 1,000, Abcam, initial raw data processing was carried out as described (9). ab8898), anti-unmodified H3 (1 in 5,000, Abcam, ab1791), or anti-β-actin (1 in 10,000; Sigma, A5441).
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