GSM9825774: GFP+; mApple- rep2, GCP; Mus musculus; RNA-Seq
After the removal of the meninges from the cerebellar tissue, it was sectioned into small pieces approximately 0.5 mm in size. Subsequently, these fragments were digested with a cold protease cocktail solution, which contained 10 mg/ml protease from bacillus licheniformis (Sigma-Aldrich) and 2.25 mg/ml deoxyribonuclease I from bovine pancreas (Sigma-Aldrich), 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. A total of 1,000 Atoh1-GFP-positive GCPs were isolated from Sin3ACKO and Control mice to generated RNA-seq sequencing libraries. Cellular RNA was extracted and cDNA was generated using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takarabio) according to manufacturer's instructions. NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) was utilized to construct libraries from cDNA. All libraries are sequenced and performed at multiplex Paired-End 150 bp on an Illumina NovaSeq 600 at Vanderbilt Technologies for Advanced Genomics.