Instrument: Illumina HiSeq 2000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: Crosslinked samples were resuspended in FA buffer, and sonicated in a Diagenode Bioruptor (60 pulses of 30 seconds at full power with 1 minute rest in between). Extracts were clarified, protein concentrations determined using a Qubit fluorometer, and precleared with Protein A Dynabeads before proceeding to ChIP. DRM subunits were individually IPed using 1-5 μg appropriate antibodies on prepared lysates. ChIPs were performed with 1-2 mg of extract, and 2% of the extract was set aside for an input reference control. ChIPs were incubated overnight at 4°C with 1% sarkosyl. Protein A Dynabeads equilibrated in 20 μL FA buffer were added and incubated for 2 hours at 4°C. ChIPs were washed with the following buffers: once with FA buffer containing 1 M NaCl, once with FA buffer containing 0.5 M NaCl, once with TEL buffer (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA), and twice with TE buffer (10 mM Tris-HCl pH 8.0 and 1 mM EDTA). 2 elutions of 50 μL elution buffer containing TE plus 1% SDS and 250 mM NaCl were incubated at 55°C. Eluted ChIP and input samples were incubated with proteinase K for 1 hour at 55°C. Crosslinks were reversed overnight at 65°C. DNA was purified by phenol-chloroform extraction and ethanol precipitation using glycogen as a carrier. Libraries from wild-type late embryos, extracts rep 1a, 1b, 2a, and 2b: Both ChIP and input DNA samples have their ends blunted and phosphorylated with KlenowDNAP, T4 DNAP, and T4 PNK. 3?-dA overhangs are then added with Klenow Fragment (3? to 5?exo minus), and Illumina adapters are subsequently ligated to the ends. Next, PCRamplification is performed using Illumina 1.1 and 2.1 primers, for a total of 18 cycles. AmplifiedDNA libraries are size-selected on a 2% agarose gel so that fragments sized between 250-350bp are obtained; gel extraction is carried out at room temperature. Libaries from wild-type late embryo extract rep 3 and lin-35(n745) late embryo extracts rep 1-3 were prepared using the TruSeq ChIP Sample Prep Kit (Illumina). Amplifed libraries were size selected to obtain 200-500 base pair fragments using Agencourt AMPure XP beads (Beckman Coulter).