title: GSM4876564: RING_H3K4me3_1; Mus musculus; OTHER
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. HiChIP was performed as described by Mumbach et al. 2016 Nat Methods with some modifications. We generally replaced the ChIP protocol with the one described above, we cut and ligated overnight (NEB T4 Ligase, instead of Invitrogen T4) and DNA was extracted with phenol-chlorophorm not a kit. For low cell numbers, we increased the centrifugation time to 30 minutes and 15 minutes after lysis, as well as 30 minutes after ligation to see a pellet more accurately. Generally 12 cycles were used for Tn5 Nextera PCR amplification (Illumina Nextera DNA UD Indexes Kit). We aimed for 100M read pairs for each run on a NovaSeq 6000 sequencer (Illumina).