title: GSM9054817: Molm-13, VTP50469, MLL IP; Homo sapiens; ChIP-Seq
library_construction_protocol: Crosslinked cells were lysed in shearing buffer (50mM Tris pH 8.0, 1% (v/v) SDS, 10mM EDTA supplemented with protease and phosphatase inhibitors). Chromatin was sonicated to 250-1000 bp fragments using the Covaris ME220 Focused-ultrasonicator. Debris was cleared by retaining the supernatant after centrifugation at 20,000 x g for 15 min at 10 C. MLL1 and Menin immunoprecipitations were performed from 50 ug and 20 ug of sonicated chromatin respectively following quantification by Quibit DNA BR quantification kit. Chromatin was diluted with wash buffer (50mM Tris pH 8.0, 150mM NaCl, 2mM EDTA, 1% v/v Triton X-100, supplemented with protease inhibitors) and immunoprecipitation overnight at 4oC with rotation mixing. Antibody bound complexes were immobilised with 30μL of pre-washed Protein A Dynabeads (Thermofisher) for 4 hours at 4oC with rotation mixing and collected using a magnetic rack. Beads were washed five times with wash buffer, once with high-salt wash buffer (wash buffer with 500mM NaCl and 0.1% SDS)  followed by a final wash with TE buffer (10mM Tris pH 8.0, 1mM EDTA). Crosslinks were reversed by incubating beads in elution buffer (20mM Tris pH 7.5, 200mM NaCl, 5mM EDTA, 1% v/v SDS, 200μg/mL Proteinase K (Roche) and 200ug/mL RNAse A (Qiagen) at 65 C overnight with gentle agitation (1000 rpm). Immunoprecipitated DNA was purified using Qiagen MinElute columns (Qiagen). ChIP-seq libraries were prepared using the Takara Bio Thruplex DNA-Seq Kit as per manufacturers instructions. Samples were sequened