GSM5952006: DIPG13-KO_EZH2; Homo sapiens; OTHER
CUT&RUN assay was done as described in Skene et al (2017) with slight modifications as follows. Cells were harvested and counted with 200000 cells per reaction. Permeabilized cells, bound to Concanavalin A-coated beads were mixed with individual primary antibody (see antibody list) and incubated overnight at 4°C while rotating. Secondary antibody, anti-rabbit HRP was used as a negative control. pAG-MNase enzyme was added to each sample followed by incubation step of 1 hour at 4°C. Targeted digestion was done by 15 minutes incubation on ice block (0°C) under low salt conditions. DNA purification was done using Nucleospin gel and PCR clean-up kit (Machery-Nagel, 740609). Libraries were prepared from 1-20ng of DNA as previously described by Blecher-Gonen et al (2013). Paired end sequencing was done on a Next-Seq 500 instrument (Illumina) using a V2 150 cycles mid output kit.