Antibody against h3k9me3

HeLa cells were transfected with 500ng each of Cas9 alone vector versus Cas9 vectors encoding sgRNAs targeting TARDBP as described above. Cells were puromycin selected for two days and allowed to recover until nine days post-transfection. Cells were cross-linked with 1% formaldehyde at 37 degrees for 10 minutes followed by quenching with 125uM Glycine. Cells were lysed (0.5% SDS, 10mM EDTA, 50mM Tris/HCl, pH 8) and sonicated on high power for 30 cycles using a Bioruptor 300 (Diagenode, Denville, NJ). Five mg of chromatin was incubated overnight at four degrees with Protein A Dynabeads (Life Technologies) and 10 mL of antibody against H3K9me3 (Cell Signaling #13969) or non-specific IgG. Beads were washed with buffers of increasing stringency (low salt, high salt, lithium chloride and TE buffer) and DNA was eluted in buffer containing 1% SDS. RNA and protein were digested using RNase A (Thermo Scientific) and proteinase K (Promega). DNA was purified using a PCR purification kit (QIAGEN) and two ml of a 1:1000 dilution was input into a Taqman qPCR reaction as described above. Percentage of input was calculated and normalized to control (Cas9 alone) samples.