H3K4me3-sgFMO3-1
For ChIP assays, cells were grown to 80% confluence and fixed with 1% formaldehyde for 10 minutes. ChIP was performed with ChIP-grade anti-H3K4me3 antibodies according to standard methods (chromatin shearing by sonication with Bioruptor Pico; Diagenode). After washes, DNA-protein complexes immobilized to Protein A/G agarose beads (Santa Cruz Biotechnologies, Inc.) were processed according to the traditional method (see below) or the ChIP Elute Kit.Traditional ChIP DNA recovery method: DNA-protein complexes were eluted from the beads in SDS buffer (1% SDS, 0.1 M NaHCO3) then DNA-protein cross-links were reversed in the presence of 250 mM NaCl at 65C overnight. Finally, a Proteinase K treatment was performed to digest proteins. After elution and cross-linking reversal, the DNA was purified and concentrated with a Macherey-Nagel NucleoSpin Gel and PCR Clean-Up kit (using the NTB buffer). Samples processed with ChIP Elute Kit were handled according to the kit protocol. The ChIP Elute Kit includes DNA clean-up and concentration as part of the protocol, additional purification prior to sequencing library preparation is not necessary.For a direct comparison between the ChIP Elute Kit and the traditional method, beads with bound complexes were split after the last wash step, and half of the material was processed with the traditional method, while the other half was processed with the ChIP Elute Kit. Sequencing libraries were generated using the DNA SMART ChIP-seq kit and size selection was performed with AMPure XP beads (Beckman Coulter) using Option 1 or Option 4 as described in the DNA SMART ChIP-seq kit User Manual.