title: GSM4876533: EED_H3K4me3_2; Mus musculus; ChIP-Seq
library_construction_protocol: Fertilized chicken eggs (white leghorn; Gallus gallus domesticus) were obtained from a local breeder (LSL Rhein-Main) and incubated at 37°C and 80% humidity in a normal poultry egg incubator (Typenreihe Thermo-de-Lux). Following microsurgical procedures, the eggs were re-incubated until the embryos reached the desired developmental stages. The developmental progress was determined according to the staging system of HH (Hamburger and Hamilton, 1992). Mice were housed and bred under standard conditions in the CECAD ivRF. The breedings described were approved by the Landesamt für Natur, Umwelt, und Verbraucherschutz Nordrhein-Westfalen (LANUV), Germany (animal application 84-02.04.2015.A405). Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibodies againts the specific proteins of interest in each case. Cells were crosslinked in 1% of formaldehyde for 20 minutes (rotating at RT) with subsequent quenching by glycine (0.125M; rotating at RT). Washed twice with PBS and 25x Roche protease inhibitor. Then flash frozen in liquid nitrogen and stored at -80°C. Afterwards cells were thawed for ~30 minutes and lysed for 10 minutes at 4°C rotating in 50 mM Hepes, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% TX-100 together with protease inhibitor (Lysis Buffer 1). After centrifugation for 5 min at 2000rcf at 4°C and discarding the supernatant, pellet was resuspended in 10 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, protease inhibitor (Lysis Buffer 2) and lysed for 10 minutes at 4°C rotating. After centrifugation for 5 min at 2000rcf at 4°C and discarding the supernatant, pellet was resuspended in 10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate and 0.5% N-lauroylsarcosine with protease inhibitor (Lysis Buffer 3). Cells were then sonicated with ActiveMotif Sonicatior (amplitude: 25%, on=20s, off=30s, 20 cycles). Debris was then centrifugated for 10 minutes, 16000rcf at 4°C. Supernatant was resuspended with 10% Triton X-100 and 10% of mixture taken for input sample. 5 ul antibody (1 ng/ul) was added to sonicated chromatin aliquot for each ChIP reaction and inverted to mix. Then rotated vertically at 4°C overnight (12-16 hrs) to bind antibody to chromatin. On the next day, 50ul (for low cell numbers) or 75ul (for higher cell numbers) of magnetic Dynabeads (Protein G) were washed three times (3X) in 1 mL cold Block Solution (0.5% BSA (w/v), 1x PBS). Antibody-bound chromatin was added to beads and inverted to mix. Then rotated vertically at 4°C for at least 4 hrs. Afterwards bound beads were washed 5X in 1 mL cold RIPA buffer (50 mM Hepes, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate). Then washed once in 1 mL TE + 50 mM NaCl on ice and centrifugated for 3 minutes at 1000rcf at 4°C to remove all remaining TE. 210 uL Elution Buffer was added to beads and DNA was eluted for 15 min at 65°C, 900 RPM, before centrifugating beads 1 min at 16000rcf at RT, adding to magnet and transferring supernatant (~200 uL) to fresh microfuge tube once beads settled. Both sample and input were then reverse crosslinked in appropriate amount of TE wash buffer, 20 mg/mL of RNAse, then Proteinase K, before extracting the DNA by phenol-chlorophorm. DNA content was measured using Qubit and the HS DNA Kit. Enrichment was measured using technical triplicates (input: 1%) by ChIP-qPCR with custom-made primers (Suppl.) on a Light Cycler 480II (Roche). All antibodies used in this study have been previously reported as suitable for ChIP: H3K4me3 (39159, Active Motif), H3K27ac (39133, Active Motif), H3K27me3 (39155, Active Motif).