GSM3262784: KMT2D Mutant H3K27me3_ChIPseq; Mus musculus; ChIP-Seq
Crosslinked KMT2D WT and Mutant samples were thawed on ice and resuspended in RIPA lysis buffer supplemented with protease inhibitor, lysed the cells on ice then sheared the chromatin by sonication. After centrifuge, the clean sonicated cell lysate mixed with protein G magnetic dynabeads (Invitrogen) coupled to antibodies described above and incubated over night at 4 degrees. Samples was washed 5 times with cold RIPA, twice with high salt RIPA, twice with LiCl buffer, twice with TE, and then eluted in elution buffer. The eluate was reverse crosslinked at 65C. Solid-phase reversible immobilization (SPRI) cleanup steps were performed. Beads were washed twice on the magnet with 70% ethanol and then air-dried. The DNA was eluted in EB buffer. Libraries for Illumina sequencing were generated following the New England BioLabs NEBNext Ultra DNA Library Prep Kit protocol. A total of 10x cycles were used during PCR amplification for the generation of all ChIP-seq libraries. Amplified ChIP DNA was purified using double-sided AMPure XP to retain fragments ~200 to 500bp and quantified using the Qubit 2000 and Bioanalyzer 1000 before multiplexing.
GEO Accession
GSM3262784