anti-DOT1L (Cell signaling #77087S clone 1W4Z lot 0)
Sequenced DNA Library
The cells were collected and washed twice in ice-cold PBS followed by crosslinking in 1% formaldehyde (methanol free) for 5 minutes. Excess of formaldehyde was quenched by addition of Tris pH 8.0 to 100nM final and Glycine 125 mM final. Chromatin was sheared using Covaris E220 for 20 min with power 140; duty 5; bursts 200/sec. We then used 5ul aliquot of sheared chromatin to decrosslink in order to test completeness of shearing. Chromatin was considered passed QC if fragments 100-600bp were >90%. For immunoprecipitation we used approximately chromatin from 1-10x10^6 cells, 5-10ug of specific antibody and 10-20ul of Protein-A/G magnetic beads (Dynal). In 16 hours the immune complexes were washed in Mixed Micelle Buffer, buffer 500, LiCl/detergent solution and TE buffers. DNA was eluted from the beads using 100mM NaHCO3 100mM NaCl 1% SDS and purified using AmpureXP beads. 1-10 ng of ChIP'ed DNA was used for Illumina compatible library generation using ThruPlex DNA-seq kit with 10 to 14 cycles of amplification with single or paired end barcodes.