GSM6387587: gastru_merged_K36-K9m3; Mus musculus; OTHER
Four to five embryos were pooled for each reported time (E9.5, E10.5, E11.5) point prior to single cell isolation. Pooled embryos were dissociated in TrypleE for 10min at room temperature. Undigested portions were physically removed and the remainder filtered through a 30 micrometer filter before the single cell suspension was split into 3 samples for each timepoint and each scChiX experiment. Per timepoint, two single cell samples were used each for a single antibody incubation (H3K36me3 or H3K9me3) and one sample for the double antibody incubation (H3K36me3 + H3K9me3). Antibody incubation was performed as described in the scChiX protocol prior to single cell capture using flow cytometry. DNA library was prepared for each sample using the scChiX protocol. Cells were processed in 0.5 mL protein low-binding tubes. Following steps are performed on ice. Cells were resuspended in 500 microliter Wash Buffer 47.5 mL H2O RNAse free,  1 mL 1 M HEPES pH 7.5 (Invitrogen),  1.5 mL 5M NaCl, 3.6 mL pure spermidine solution (Sigma Aldrich)).     Cells were pelleted at 600 gram for 3 minutes and resuspended in  400 microliter Wash Buffer 1 (wash buffer with 0.05 % saponin (Sigma Aldrich), protease inhibitor cocktail (Sigma Aldrich), 4 microliter 0.5M EDTA) containing the primary antibody (1:100 dilution for H3K4me1, H3K27me3 and H3K4me1+H3K27me3 Saponin has to be prepared fresh every time as a 10% solution in PBS). Cells were incubated overnight at 4 degrees Celsius on a roller, before they were washed once with 500 microliter Wash Buffer 2 (wash buffer with 0.05 percent saponin, protease inhibitor). Afterwards cells were resuspended in 500 microliter Wash Buffer 2 containing Protein A-Micrococcal Nuclease (pA-MNase) 3 nanogram per milliliter and incubated for 1 hour at 4 degrees Celsius on a roller.    Finally, cells were washed an additional 2 times with 500 microliter Wash Buffer 2 before passing it through a 70 micrometer cell strainer (Corning, 431751) and sorting G1 cells based on Hoechst staining on a BD Influx FACS machine into 384 well plates containing 50 nanoliter Wash buffer 3 (Wash buffer containing 0.05% saponin) and 5 microliter sterile filtered mineral oil (Sigma Aldrich) per well. Small volumes were distributed using a Nanodrop II system (Innovadyme).