Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: After crosslinking with 1% formaldehyde for 10 minutes, chromatin shearing was done by sonication in a Bioruptor UCD-200 (Diagenode, Liège, Belgium). The resulting lysate was divided into one aliquot for streptavidin capture (the equivalent of 9-10 million cells), another for Histone 3 K4 tri-methylation (H3K4me3; ab 8580, Abcam, Cambridge, UK) immunoprecipitation (the equivalent of 1-2 million cells) and a third aliquot for non-immunoprecipitated lysate to use as input control. The H3K4me3 immunoprecipitation and the input control preparation were done using the MagnaChip kit, according to the manufacturer’s instructions, increasing the reaction volumes to accommodate the input cell number. For the streptavidin capture, Dynabeads M-280 Streptavidin (Invitrogen) were used, according to the manufacturer’s recommendations with minor modifications. Briefly, beads were washed with PBS three times prior to use, 3 mg of beads were used for each sample, incubation was done at room temperature, and washing after incubation was done five times with PBS with 0.1% BSA. After washing, the streptavidin beads were resuspended in Chip Elution Buffer from the MagnaChip kit and all samples proceeded to Proteinase K treatment and DNA purification according to the MagnaChip protocol. ThruPlex DNA-seq kit (Rubicon Genomics, Ann Arbor, Michigan, USA) according to manufacturer’s recommendations.