title: GSM7350372: Input; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP experiments were carried out using ChIP Reagents (Nippon Gene). ChIP was performed in E15.5 WT and Nsd2 KO brain. Fresh brain tissue was minced and fixed with 1% paraformaldehyde in D-PBS (-) for 10 min at room temperature and quenched with 2 M glycine for 5 min. Tissue was resuspended in NP-40 buffer containing proteinase inhibitor cocktail (Nacalai Tesque) and homogenated by BioMAsher (Nippi). Homogenized tissue was incubated for 10 min on ice and centrifuged to collect nuclear pellets. Nuclear pellet was resuspended in SDS Lysis buffer and lysate was sonicated to fragment chromatin for 15 min (Duty:5, PIP:105, cycles/burst:200) in Covaris S220 (Covaris). The fragmented chromatin was centrifuged to remove debris and immunoprecipitated with Dynabeads Protein A (Thermo Fisher) conjugated anti-H3K36me2 (ab9049, Abcam) in ChIP dilution buffer with protease inhibitor cocktail for 4 hr at 4℃ rotating. The beads were washed with 1 x RIPA buffer - 150 mM, 1 x RIPA buffer - 500 mM, LiCl buffer (250 mM LiCl, 10 mM Tris-HCl(pH8.0), 0.5% NP- 40, 0.5% Sodium deoxycholate, 1 mM EDTA) and TE buffer (pH 8.0). The beads were incubated in ChIP direct elution buffer with Proteinase K at 65 ℃ overnight. ChIP DNA was purified using AMPure XP beads (Beckman Coluter) following the manufacturer's protocol. ChIP-seq libraries were generated using NEBNext Ultra Ⅱ DNA Library prep Kit for Illumina (New England Biolabs) following the manufacturer's protocol.