title: GSM6500399: neurospheres, CBPf/f; Mus musculus; RNA-Seq
library_construction_protocol: For the single nucleus RNA-Seq experiment, a pool of ~15 differentiated neurosphere cultures from control, CBPf/f+Cre and p300f/f+Cre at 15DIV were used. Differentiation medium II was removed and wells were washed with cold HBSS. Cold Lysis Buffer (10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2, and 0.1% NonidetTM P40 Substitute in Nuclease-Free Water) was added (500 µl) immediately to every well, gently pipetted until cells were completely suspended and kept on ice for 5 min to lyse cells. The nuclei suspension from the different samples (control, CBPf/f+Cre and P300f/f+Cre) was transferred to a 15ml tube and centrifuged at 500 rcf for 5 min at 4°C. Supernatant were removed and nuclei pellet was resuspended in 1 ml of Nuclei Wash and Resuspension Buffer (1X PBS with 1.0% BSA and 0.2 U/μl RNase Inhibitor). Centrifugation and nuclei wash steps were repeated twice. Nuclei suspension was sorted on FACS Aria III (BD Biosciences) at 4°C and immediately proceed to 10x Genomics single cell protocol. 16,000 nuclei per sample (pool of ~15 differentiated neurosphere cultures) were loaded into single cell Chromium Chip B and Chromium Controller was run to generate partitioning thousands of nuclei into nanoliter-scale Gel Beads-in-emulsion (GEMs) and immediately reversed transcribed, where all generated full-length cDNA (from poly-adenylated mRNA) share a common 10x Barcode. Post-GEM-RT cleanup, cDNA amplification and 3ʹ Gene Expression Library Construction were performed with Chromium Single Cell 3' Library & Gel Bead Kit v3. Quality control of cDNA and libraries were performed with Agilent Bioanalyzer in a High Sensitivity chip.