Ez lysis buffer

Ultra-low input ChIP-seq was performed as previously described^{45 (link)}, incorporating several adaptions from the original protocol^{46 (link)}. In brief, replicates of ExE samples were permeabilized in nuclei EZ lysis buffer (Sigma) with 0.1% Triton-X-100/0.1% deoxycholate. Chromatin digestion was completed with 200 U of micrococcal nuclease (New England Biolabs) at 21 °C for 7.5 min. Chromatin was then precleared in complete immunoprecipitation buffer with Protein A/G beads, for 2 h rotating at 4 °C. Chromatin was then divided into three aliquots: one for each immunoprecipitation and one 10% input control. Antibodies for H3K4me1 (250 ng, Active Motif, 39298) or H3K27ac (125 ng, Abcam, ab4729) were bound to Protein A/G beads in complete immunoprecipitation buffer for 3 h rotating at 4 °C. Chromatin was added to antibody-bound beads and rotated overnight at 4 °C. Chromatin-bound beads were washed with two low-salt washes and one high-salt wash, followed by DNA elution at 65 °C for 90 mins. Eluted DNA was purified with Sera-Mag carboxylate-modified Magnetic SpeedBeads (Fisher Scientific) at a 1.8:1 ratio. Library preparation and indexing was performed using the MicroPlex Library Preparation kit v2 (Diagenode), as per the manufacturer’s instructions. Libraries were multiplexed for 75-bp paired-end sequencing on an Illumina NextSeq500.

Whole-cell extract preparation was performed using lysis buffer (50 mM Hepes, pH 7.4, 150 mM NaCl, 15 mM MgCl₂, 10% glycerol, phosphatase and protease inhibitor cocktails (Roche, Basel, Switzerland), 0.5% Triton X-100). After 10 min incubation on ice, the cell suspension was centrifuged for 10 min at 12,000× g at 4 °C, and the supernatant fractions were collected and used for further analyses.
Cell fractionation was obtained through two consecutive extractions. First, nuclei isolation was performed using ice-cold nuclei EZ lysis Buffer (Sigma Aldrich) according to the manufacturer’s instructions. Second, chromatin-bound protein fraction was isolated by diluting the nuclei pellet in ice-cold extraction buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM EDTA supplemented with phosphatase and protease inhibitor cocktails) containing 200 μg/mL RNase A and incubating for 30 min at 25 °C under agitation. Following centrifugation at 14,000× g for 3 min, the pellet was resuspended in PBS buffer supplemented with 1% SDS, heated for 10 min at 100 °C and sonicated for 10 s. Concentrated loading sample buffer was added for a 1× final concentration in all protein lysates, and the samples were boiled for 5 min. Western blot analysis was carried using the antibodies listed in Table 1.

Day 6 uteri from the control and Ezh2 uKO were used. Uterine tissues surrounding the embryos were longitudinally opened at the mesometrial side and kept at −80 °C until use. Endometrial tissues were disrupted with 35 strokes in a Dounce homogenizer on ice, with a loose-fitting pestle in PBS-containing protease inhibitor cocktail and phosphatase inhibitors 2 and 3 (Sigma, St. Louis, MO, USA). After centrifugation with 1000g for 5 min, pellets were received in the nuclei EZ lysis buffer (Sigma) to isolate the nuclei. Native ChIP was then performed as previously described [63 (link)], with some modifications. For the fragmentation, chromatins were treated by 20 U/μl MNase at 37 °C for 5 min. Input DNA was analyzed with a 2100 Bioanalyzer system using High-Sensitivity DNA Reagent kit (Agilent, Santa Clara, CA, USA) to confirm DNA fragmentations at approximately 200–300 bps. Chromatin immunoprecipitation was performed using Magna ChIP G-Chromatin Immunoprecipitation Kit (Millipore, Burlington, MA, USA) following the manufacturer’s protocol. Anti-H3K27me3 antibody (39155, Active motif, Carlsbad, CA, USA) or anti-rabbit IgG (2729, Cell Signaling Technology) was used to precipitate the target chromatins. Chromatin-immunoprecipitated DNAs were purified by extracting phenol–chloroforms.