SRX5226971 GSM3554032 SRP176629 SRS4230823 GSM3554032 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554032 GSM3554032 GEO Accession GSM3554032 SRX5226972 GSM3554033 SRP176629 SRS4230824 GSM3554033 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554033 GSM3554033 GEO Accession GSM3554033 SRX5226973 GSM3554034 SRP176629 SRS4230825 GSM3554034 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554034 GSM3554034 GEO Accession GSM3554034 SRX5226974 GSM3554035 SRP176629 SRS4230826 GSM3554035 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554035 GSM3554035 GEO Accession GSM3554035 SRX5226975 GSM3554036 SRP176629 SRS4230827 GSM3554036 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554036 GSM3554036 GEO Accession GSM3554036 SRX5226976 GSM3554037 SRP176629 SRS4230828 GSM3554037 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554037 GSM3554037 GEO Accession GSM3554037 SRX5226977 GSM3554038 SRP176629 SRS4230829 GSM3554038 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554038 GSM3554038 GEO Accession GSM3554038 SRX5226978 GSM3554039 SRP176629 SRS4230830 GSM3554039 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554039 GSM3554039 GEO Accession GSM3554039 SRX5226979 GSM3554040 SRP176629 SRS4230831 GSM3554040 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554040 GSM3554040 GEO Accession GSM3554040 SRX5226980 GSM3554041 SRP176629 SRS4230832 GSM3554041 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554041 GSM3554041 GEO Accession GSM3554041 SRX5226981 GSM3554042 SRP176629 SRS4230833 GSM3554042 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554042 GSM3554042 GEO Accession GSM3554042 SRX5226982 GSM3554043 SRP176629 SRS4230834 GSM3554043 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554043 GSM3554043 GEO Accession GSM3554043 SRX5226983 GSM3554044 SRP176629 SRS4230835 GSM3554044 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554044 GSM3554044 GEO Accession GSM3554044 SRX5226984 GSM3554045 SRP176629 SRS4230836 GSM3554045 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554045 GSM3554045 GEO Accession GSM3554045 SRX5226985 GSM3554046 SRP176629 SRS4230837 GSM3554046 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554046 GSM3554046 GEO Accession GSM3554046 SRX5226986 GSM3554047 SRP176629 SRS4230838 GSM3554047 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554047 GSM3554047 GEO Accession GSM3554047 SRX5226987 GSM3554048 SRP176629 SRS4230839 GSM3554048 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554048 GSM3554048 GEO Accession GSM3554048 SRX5226988 GSM3554049 SRP176629 SRS4230840 GSM3554049 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554049 GSM3554049 GEO Accession GSM3554049 SRX5226989 GSM3554050 SRP176629 SRS4230841 GSM3554050 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554050 GSM3554050 GEO Accession GSM3554050 SRX5226990 GSM3554051 SRP176629 SRS4230842 GSM3554051 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554051 GSM3554051 GEO Accession GSM3554051 SRX5226991 GSM3554052 SRP176629 SRS4230843 GSM3554052 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554052 GSM3554052 GEO Accession GSM3554052 SRX5226992 GSM3554053 SRP176629 SRS4230844 GSM3554053 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554053 GSM3554053 GEO Accession GSM3554053 SRX5226993 GSM3554054 SRP176629 SRS4230845 GSM3554054 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554054 GSM3554054 GEO Accession GSM3554054 SRX5226994 GSM3554055 SRP176629 SRS4230846 GSM3554055 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554055 GSM3554055 GEO Accession GSM3554055 SRX5226995 GSM3554056 SRP176629 SRS4230847 GSM3554056 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554056 GSM3554056 GEO Accession GSM3554056 SRX5226996 GSM3554057 SRP176629 SRS4230848 GSM3554057 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554057 GSM3554057 GEO Accession GSM3554057 SRX5226997 GSM3554058 SRP176629 SRS4230849 GSM3554058 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554058 GSM3554058 GEO Accession GSM3554058 SRX5226998 GSM3554059 SRP176629 SRS4230850 GSM3554059 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554059 GSM3554059 GEO Accession GSM3554059 SRX5226999 GSM3554060 SRP176629 SRS4230851 GSM3554060 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554060 GSM3554060 GEO Accession GSM3554060 SRX5227000 GSM3554061 SRP176629 SRS4230852 GSM3554061 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554061 GSM3554061 GEO Accession GSM3554061 SRX5227001 GSM3554062 SRP176629 SRS4230853 GSM3554062 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554062 GSM3554062 GEO Accession GSM3554062 SRX5227002 GSM3554063 SRP176629 SRS4230854 GSM3554063 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554063 GSM3554063 GEO Accession GSM3554063 SRX5227003 GSM3554064 SRP176629 SRS4230855 GSM3554064 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554064 GSM3554064 GEO Accession GSM3554064 SRX5227004 GSM3554065 SRP176629 SRS4230856 GSM3554065 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). NextSeq 500 gds 303554065 GSM3554065 GEO Accession GSM3554065 SRX5227005 GSM3554066 SRP176629 SRS4230857 GSM3554066 ChIP-Seq GENOMIC ChIP 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). NextSeq 500 gds 303554066 GSM3554066 GEO Accession GSM3554066 SRX5227006 GSM3554067 SRP176629 SRS4230858 GSM3554067 ChIP-Seq GENOMIC ChIP 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). NextSeq 500 gds 303554067 GSM3554067 GEO Accession GSM3554067 SRX5227007 GSM3554068 SRP176629 SRS4230859 GSM3554068 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). Illumina MiSeq gds 303554068 GSM3554068 GEO Accession GSM3554068 SRX5227008 GSM3554069 SRP176629 SRS4230860 GSM3554069 ChIP-Seq GENOMIC ChIP The larvae were collected and rinsed with PBST (PBS + 0.1% Triton X-100) three times to remove adhering corn food. One volume of hexanes (a mixture of isomers) was equilibrated for 30 min against 0.175 vol of 37% formaldehyde and 0.130 vol of 10X PBS (1.37 M NaCl, 27 mM KCl, 43 mM Na2HPO4 and 14 mM KH2PO4), pH 7.5, to make the mixture 5% formaldehyde and 1X PBS. Only the upper organic phase was used for crosslinking. Larvae were fixed in 10 ml of buffered 5% formaldehyde/hexanes per gram of larvae by vigorous shaking for 5 min at room temperature. The larvae were allowed to settle, and the formaldehyde/hexanes solution was removed by centrifugation (3,000 x g for 5 min at RT). The larvae were washed twice in a solution containing 1X PBS and 0.5% Triton X- 100 (0.5% PBST), using the five volumes as used for fixing. They were then stored at -80°C. Chromatin was purified by quickly thawing frozen larvae and resuspending them in buffer A [0.3 M sucrose, 15 mM NaCl, 5 mM MgCl2, 15 mM Tris pH 7.5, 60 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 0.5 mM dithiothreitol (DTT) and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. They were homogenized with 20-25 strokes of a hand-held type B Dounce homogenizer. Triton X-100 was added to 0.3%, and the homogenate was centrifuged (2,000 x g for 15 min at 4°C). The pellet was resuspended in buffer B (100 mM NaCl, 10 mM Tris pH 7.9, 1 mM EDTA, 0.1% v/v NP-40 and 1 mM PMSF) and homogenized with ~5 strokes of a hand-held type B Dounce homogenizer. The homogenate was then sheared by 20 cycles (20 × 20 s on and 50 s off cycles, 40% power settings) through a sonicator (Sonic Dismembrator Model 500, Fisher Scientific) to an average size of ~200 to 700 bp chromatin fragments. After sonication, debris was removed by centrifugation at 4°C for 10 min at 13,000 r.p.m., and an equal amount of 6 M urea was added to the solution and then incubated for 10 min at 4°C on a rotating wheel. The soluble chromatin was dialyzed 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 r.p.m., 2 min). The chromatin solution was pre-cleared with 100 μl Streptavidin-agarose slurry (Sigma) beads for O-GlcNAz modified or vehicle-only chromatin separately and incubated for 1 hour at 4°C on a rotating wheel. The pre-cleared chromatin was aliquoted after centrifugation at 4°C for 2 min at 7,500 r.p.m., snap-frozen in liquid nitrogen and stored at −80°C for future use. For click chemistry, 20-50 μg chromatin DNA was incubated with iodoacetamide (IAA) to a final concentration of 15 mM, agitate mildly for 30 min at RT then added DMSO solution of dibenzylcyclooctyne-S-S-PEG3-Biotin (DBCO-S-S-PEG3-Biotin, Jena Bioscience GmbH) to a final concentration of 40 μM. The samples were protected from light and agitated mildly for 1 hour at RT. The unreacted probe was filtered off by the Amicon Ultra-0.5 mL centrifugal filter (15K cut-off) (EMD Millipore). Biotinylated-probe-chromatin complexes were captured by incubation with 50 μl Streptavidin magnetic beads (NEB) at 4°C for 2 hours. Beads were washed with 0.5 ml of the following buffers: three washes with low-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 150 mM NaCl), followed by three washes with high-salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.0, 500 mM NaCl), then three washes with lithium wash buffer (0.25 M LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl pH 8.0); then two washes with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Beads were resuspended in 100 μl TE containing 0.5% SDS, 15 mM DTT and incubated with 0.15 mg/ml proteinase K at 55°C for 3-4 hours. DNA-protein complex was de-crosslinked by incubating overnight at 65°C (not more than 15 hours). ChIP-DNA was purified with QIAquick PCR Purification Kit and stored at -20°C. 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). Illumina MiSeq gds 303554069 GSM3554069 GEO Accession GSM3554069