Lysates were clarified from sonicated nuclei and OGA/DNA complexes were isolated with MEGA5 antibody (Proteintech #14711-1-AP). Proc. Natl. Acad. Sci. U.S.A. 2014, 111, 5592-5597.
Sequenced DNA Library
50 larvae were cross-linked in 5% formaldehyde in PBS with 0.5% Triton X-100 (0.5% PBST) for 5 min by vortexing occasionally at room temperature. The flies were then washed three times in 0.1% PBST, using the five volumes as used for fixing. and glycine was added to a final concentration of 0.125 M. The samples were washed once with PBS, 1 mM PMSF, protease inhibitor cocktail (Roche) followed by two washes in PBS, 1 mM PMSF and protease inhibitor cocktail. These flies were homogenized in 500 μl Lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.1). To ensure shearing of the cross-linked DNA into 200-500 bp long fragments, 120 μl glass beads were added prior to sonication for 4 min using a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) at high setting (30 s ON, 30 s OFF). The samples were cleared by centrifuging for 10 min at 14,000 x g at 4 °C. For each ChIP (WT and OGA-null), 150 μl of cell lysate was diluted by a factor of ten in ChIP Dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH 8.0, 167 mM NaCl), and protein inhibitors were added. To reduce nonspecific background the diluted lysate was pre-cleared by incubation with 60 μl of equilibrated Dynabeads Protein A (Thermo Fisher Scientific) for 30 min at 4 °C with agitation. For immunoprecipitation, the cleared lysates were incubated with 5 μl of normal rabbit IgG (Control; Santa Cruz, sc-2027) and rabbit antibodies (MEGA5, Proteintech #14711-1-AP) raised against full-length Drosophila OGA protein overnight at 4 °C on a rotating platform. The antibody complexes were precipitated by incubation with equilibrated Protein A Dynabeads for 1 h at 4 °C. The beads were washed for 4 min with agitation at 4 °C with the following buffers; once with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM Tris-HCl pH 8.0, 150 mM NaCl), once with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM Tris-HCl pH 8.0, 500 mM NaCl), once with LiCl-containing buffer (250 mM LiCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate), and twice with TE Buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The protein/DNA complexes were eluted from the antibody by incubating for 15 min at room temperature in 250 μl Elution buffer (1% SDS, 0.1 M NaHCO3) with rotation. The elution was repeated once, and the eluates were combined to a total volume of 500 μl. NaCl was added to a final concentration of 200 mM, and protein/DNA crosslinks were reversed by heating at 65 °C for 4 h. A total of 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl pH 6.5, and 1 μl of 20 mg/ml proteinase K were added before an additional incubation at 45 °C for 1 h. The DNA was recovered by phenol/chloroform extraction followed by ethanol precipitation. The immunoprecipitated DNA was then dissolved in 24 μl water. 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 High Sensitivity dsDNA Agilent Bioanalyzer Chips and Quibit. WT and OGA-null O-GlcNAc-seq libraries and OGA ChIP-seq libraries were sequenced on the Illumina NextSeq 500 High Output (75 cycles, 400M reads) platform. The additional replicates for both WT and OGA-null DMSO control from an earlier pilot study were sequenced on the MiSeq platform, using v3, 150-cycle reagent kits (Illumina).