ChIP-qPCR and analysis

For cerebellar granule neurons, chromatin immunoprecipitation was performed following the protocol of EZ-ChIP (Millipore, 17-371). Briefly, cells were lysed by SDS Lysis Buffer and sonicated for 1.5 hrs (Diagenode Bioruptor) at 4°C on the high setting with 30 seconds on/off interval. 20 μl Dynabeads Protein G (ThermoFisher, 10003D) was pre-incubated with 2 μg antibodies in 1XPBS buffer for 4 to 6 hours at 4°C. Mouse anti-Cas9 (Encor MCA-3F9 and Diagenode C15200229-100) antibodies were used. Cell lysates were then incubated overnight with antibody-bead complexes at 4°C. Subsequently, the beads were washed with Low Salt Immune Complex Wash Buffer, High Salt Immune Complex Wash Buffer, LiCl Immune Complex Wash Buffer, and TE Buffer. Bound protein/DNA complexes were eluted by ChIP elution buffer and then reversed the crosslinks. Samples were treated with RNase A and Proteinase K for post-immunoprecipitation and then the DNA was purified using QIAquick PCR Purification Kit (Qiagen, 28104). ChIP primers sequences can be found in Supplemental Table 1. ChIP for Grin2c was normalized to ChIP for Gapdh in the same sample to control for concentration and sample handling.