GSM9825731: Wildtype rep1, GFP+; mApple-, Sin3a (ChIP); Mus musculus; ChIP-Seq
Cerebellar tissue  was sectioned into small pieces approximately 0.5 mm in size. Subsequently, these fragments were digested with a cold protease cocktail solution, which contained cold protease (Sigma-Aldrich) (5mg/ml) and Dnase (Sigma-Aldrich) (2.5mg/ml), for 15 minutes in a water bath maintained at 6℃, with gently pipetting to mechanically dissociate the tissues every five minutes during this period. After dissociation, the digestion enzyme mixtures were passed through a 40μm filter (Miltenyi Biotech) and washed twice by 1XPBS solution. The BD FACS Aria III cell sorter was used to purify GCPs based on the intensity of Atoh1-GFP fluorescence at the single-cell level. ChIP-seq was followed by the ChIPmentation protocol(Xu et al., 2020). In brief, purified cells were cross-linked with 1% formaldehyde/PBS solution at room temperature for 15 minutes before quenching with 250 mM glycine. For co-repressor(Sin3A and Hdac1) ChIP assays, an additional protein cross-linking step using 2mM disuccinimidyl glutarate was incorporated. Chromatin was subsequently fragmented to the size of 200~1000bp by sonication for 21 seconds using a Diagenode One device (Diagenode, NJ). Soluble chromatin was then obtained and incubated overnight at 4°C with the target antibody. Afterward, Dynabeads Protein A/G (10001D/10003D, Thermo Fisher Scientific, MA) were added for two hours to capture the antibody-chromatin complexes. Unbound chromatin were removed through multiple washes following a high-salt buffter, low-salt buffer, LiCl buffer, and 10mM Tris buffter sequence to ensure specificity of binding to the beads. Subsequently, an on-bead tagmentation step were performed using transposase Tn5 (20034197, Illumina, CA) to facilitate the ligation of sequencing adapters to the DNA. The DNA was then eluted from the beads and purified using AMPure XP Beads (A63880, Beckman Coulter, IN). Finally, the ChIP DNA was amplified to generate a sequencing library with Illumina Nextera DNA CD Indexes (20018707, Illumina, CA). All libraries are sequenced and performed at multiplex Paired-End 150 bp on a Illumina NovaSeq 600 at Vanderbilt Technologies for Advanced Genomics.