GalNAz feeding, Staudinger ligation, and streptavidin purification
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Pools of ~25 pupae were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These pupae pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min). Before each enrichment of O-GlcNAz modified nuclear proteins, 500 µL solution (1% SDS/PBS) consisting of 800-1,000 µg nuclear protein was mixed with 50 µL Streptavidin-agarose slurry (Sigma), incubated for 1 h at 4°C for preclearing. The supernatant was transferred to a new tube and reacted with 200 µM Biotin-azo-phosphine for overnight at room temperature. Unreacted probe was removed by chloroform/methanol precipitation. Air-dried protein pellets were resuspended in 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0) and enriched by Streptavidin-agarose slurry (Sigma) for a couple of hours at 4 °C with gentle rocking. The beads were sequentially washed trice with 5-10 volumes of 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0), PBS, and 1% SDS/PBS. Centrifugation of the beads between washing steps was carried out (7,500 rpm, 2 min). Bound proteins were cleaved from the beads by treating with one beads volume of elution buffer (100 mM Na2S2O4 in 1% SDS/PBS) for 30 min trice. Collect and combine the eluants. Removal of the majority of Na2S2O4 from the eluants was achieved by precipitating them with chloroform/methanol method; 10% of each eluted sample was analyzed by Immunoblot using Odyssey (LI-COR Biosciences). The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).