TBC1D3 suppression of the Histone Lysine N-methyltransferase G9a promotes the generation of neural progenitors and cortical expansion [ChIP-seq]

we conducted chromotin Immunoprecipitation sequencing (ChIP-seq) using human cerebral organoids treated with TAT-Scrambled (Short for TAT-Scr) with the sequence of YGRKKRRQRRR-FRVRYWFQGCHSEDPWR and TAT-465-481, a special peoptide designed for inhibition of TBC1D3, with the sequence of YGRKKRRQRRR-EGPWFRHYDFRQSCWVR. Briefly, tissues were fixed in 1% formaldehyde to cross-link histone and non-histone protein to DNA for 10 to 20 minutes at room temperature and the crosslink was stopped by the addition of glycine. Then cells were lysated to release nucleus and micrococcal nuclease was used to digest chromatin into DNA/protein fragments.A part of the diluted chromatin in each group was transferred into a new microfuge tube to be saved as the input sample and H3K9me2 (Cell Signaling Technology, 4658s) was used as the immunoprecipitating antibody to be added into diluted chromatin mixture. Incubated IP samples at 4°C for overnight with rotation. Added ChIP-Grade Protein G Magnetic Beads to each IP reaction and incubated for 2h at 4°C with rotation and then the chromatin from antibody/Protein G magnetic beads was eluted and reversed. DNA in the chromatin mixture was purified and sequenced.