title: GSM6927730: CnR_IgG2: Satb2 flx/flx DIV14 primary cortical cultures, 1h bic, IgG, rep2; Mus musculus; OTHER
library_construction_protocol: For CUT&RUN, nuclei of 106 cells were isolated by addition of nuclei extraction buffer (NEB, 20 mM Hepes (KOH) pH 7.9, 10 mM KCl, 0.5 mM Spermidine, 0.1 % Triton X-100, 20 % Glycerol, 1 tablet cOmplete EDTA-free Protease inhibitor). Cells were incubated at 4 °C for 15 min, lifted from the dish using a cell lifter and gently resuspended. Nuclei suspension was spun down at 600 g at 4 °C for 3 min. Pellet was resuspended in NEB and intact nuclei were counted in a Neubauer chamber. 100 000 nuclei were used for each CUT&RUN reaction. After Concavalin-A conjugated beads activation (21-1401-EPC Epicypher), nuclei were demobilized and blocked in blocking buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1 % BSA, 2 mM EDTA, 1 tablet cOmplete EDTA-free Protease inhibitor) at RT for 5 min. After washing in washing buffer (20 mM Hepes pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 0.1 % BSA, 1 tablet cOmplete EDTA-free Protease inhibitor) primary antibody was added and incubated at 4 °C for 2 h. Reaction was washed twice  before adding pAG-MNAse (15-1016-EPC Epicypher) and incubated for 1h at 4°C. Next, beads were washed twice before addition of 100 mM CaCl2 for 30 min at 0 °C in an ice water bath. 2X STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/ml RNAseA, 40 µg/ml Glycogen, 10 pg/ml E.Coli spike-in DNA) was added and beads were incubated for 20 min at 37 °C to release DNA fragments and to digest RNA. DNA was purified by Phenol-Chloroform extraction and precipitated in 95 % EtOH at -80 °C. Pellet was dissolved in TE buffer pH 8.0 (12090015, ThermoFisher) and stored at -20 °C. Libraries were prepared using the NEBNext Ultra Library prep Kit (E7645S, New England Biolabs) according to the manufacturer's protocol. Adapters were used according to the DNA input at a working concentration of 1.5 µM. Library was PCR amplified with unique indexes (E7335S, New England Biolabs) for 14 cycles. After clean-up fragment size and concentration was analyzed before sending for sequencing.