title: GSM8907925: CUT&RUN_REP1; Mus musculus; OTHER
library_construction_protocol: scRNA-seq: Following GEM recovery, cDNA was amplified and final libraries prepared according to the manufactuer's instructions (10x Genomics Chemistry, Single Cell version 3.0.1).  CUT&RUN Wild type mouse active adult hippocampal neural stem cells (AHNSCs) were cultured in DMEM/F-12 + Glutamax media supplemented with 1x N2, 1x penicillin/streptomycin (ThermoFisher Scientific, 15140), 3 ug/ml laminin (Sigma L2020), 5 ug/ml heparin (Sigma, H3393) and 20ng/ml Fibroblast Growth Factor (PeproTech, 450-33). The ASCL1 CUT & RUN samples (2 replicates) was performed with the CUT & RUN kit. Briefly, 500,000  cultured AHNSCs were collected in a 1.5 ml tube containing wash buffer. The cells were captured with ConA beads and incubated with 0.5 μg of the anti-ASCL1 monoclonal antibody overnight at 4°C in antibody buffer. After washing the unbound antibody, pAG-MNase was added and incubated for 10 min at RT. After washing, CaCl2 was added and incubated for 2 h and the reaction was stopped with the stop buffer. The protein-DNA complexes were released by incubating at 37°C  for 10 min. DNA was then purified using a DNA cleanup kit. scRNA-seq: Following GEM recovery, cDNA was amplified and final libraries prepared according to the manufactuer's instructions (10x Genomics Chemistry, Single Cell version 3.0.1). CUT&RUN Sequencing library was prepared using the NEBNext Ultra II DNA library preparation kit for Illumina specifically modified to make libraries from small DNA fragments. Briefly, end repair was conducted at 20°C for 30 min, followed by dA-tailing at 50°C for 60 min. After adapter ligation, the DNA fragments were purified by 1.75X volume of AMPure XP beads followed by 14 cycles PCR amplification with NEBNext Ultra II Q5 Master Mix. The PCR products were firstly cleaned up with 0.8X volume of AMPure XP beads. Then, a second cleaning was performed with 1.2x volume of AMPure XP beads to remove PCR products shorter than 150 base pairs. A final round of 1.2x AMPure XP beads clean-up was performed to eliminate PCR dimers. The library was sequenced on the NovaSeq 6000 instrument, PE100.