title: GSM3554046: O-GlcNAcseq_wt Drosophila larvae (biological rep 2)_150uM GalNAz media for 36h followed by 20h growth on DMSO media; Drosophila melanogaster; ChIP-Seq
library_construction_protocol: 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).
attributes.tag: GEO Accession
attributes.value: GSM3554046