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| Status |
Public on Jan 07, 2020 |
| Title |
O-GlcNAcseq_wt Drosophila larvae (biological rep 2)_150uM GalNAz media for 36h followed by 12h growth on DMSO media |
| Sample type |
SRA |
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| Source name |
O-GlcNAc-seq_wt larvae_150uM GalNAz media for 36h followed by 12h growth on DMSO media
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| Organism |
Drosophila melanogaster |
| Characteristics |
strain background: Oregon R
genotype/variation: wild type
stage/tissue: larvae
chip strategy: GalNAz feeding, strain-promoted click reaction and streptavidin purification
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| Treatment protocol |
Wild-type Oregon R (WT) Drosophila were expanded in 50 mL bottles of cornmeal molasses media at 25°C. Drosophila parents from approximately 25 of these bottles were required for each time course replicate. Time course Ac4GalNAz feeding was performed on 10 mL petri dishes. Just under 10 mL of cornmeal molasses media supplemented with Ac4GalNAz dissolved in DMSO to a final concentration of 150 uM or DMSO only in the same volume added to the Ac4GalNAz plates (control) were made. Adult Drosophila parents from the bottles were then transferred to the control plates for 2 hours (on cages) for a pre-egg laying period. This facilitated the laying of eggs that were not synchronized. These adult Drosophila were then transferred to either 150 uM Ac4GalNAz or control plates for 2 hours (enabling the laying of synchronized eggs). After a 36 h incubation period, the resulting larvae were transferred to control plates or collected and snap frozen (0 h time point). Larvae that were initially grown on control plates were collected at this time as well (no feed control). The larvae transferred from Ac4GalNAz plates to control plates were then collected after 4, 8, 12, 16, 20, 24, and 36 hours of feeding on these control plates.
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| Extracted molecule |
genomic DNA |
| Extraction 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).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
wt.r2_12
processed data file: TC-O-GlcNAc-seq-peaks.bed
O-GlcNAc-seq in wt Drosophila larvae (biological replicate 2) grown on 150uM GalNAz media for 36h followed by 12h growth on DMSO media
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| Data processing |
O-GlcNAc-seq: FASTQ sequencing files were aligned to a concatenated reference genome of D. melanogaster (dm3), S. cerevisiae (sacCer3), and the three synthetic gBlock fragments using the snap-aligner single command from SNAP version 1.0beta.18. The yeast chromosomes were re-name with the prefix "yeast" to avoid conflicts with the same chromosome names as Drosophila.
O-GlcNAc-seq: The resulting BAM-format alignment files were sorted, cleared of duplicates and indexed using the SAMtools commands sort, rmdup -s, and index, respectively, using SAMtools version 0.1.19-96b5f2294a
O-GlcNAc-seq: BDG files were generated from the BAM files using the command bedtools genomecov -ibam -bg command using bedtools v2.17.0. Yeast spike-in normalized BDG files were generated by multiplying by the fourth column, which specifies the sequencing coverage, of the BDG file by the normalization constants. These normalization constants were calculated by dividing the number of base pairs that aligned to the sacCer3 genome from the time point that had the least sacCer3 aligned base pairs by the number of base pairs that aligned to the sacCer3 reference genome from each time point. This was done within each replicate separately. The command awk -F'\t' '{$4=$4*a;print}' a=NF OFS='\t' <$.bdg> was used to do this, where NF is the normalization factor, which are specified in Table 1, and <$.bdg> is the appropriate time course input BDG file.
O-GlcNAc-seq: Peaks were called by calculating the coordinates that contained 5 times the average base pair sequencing coverage within each time course experiment. This was done for the no feed controls as well. The coordinates of base pairs that satisfied this requirement were then merged if they were within 30 base pairs of each other. This was done with the command awk '{ if( $4 > 5X) print}' <$.bdg> | cut -f 1,2,3 | bedtools merge -d 30, where 5X indicates 5 times the average base pair sequencing coverage within each time course experiment and <$.bdg> indicates the appropriate time course BDG file. For each time point, the peaks that occurred from the no feed controls were eliminated from the time course experiment peaks using the bedtools intersect -v command. This effectively removed false positives from the analysis. Peaks resulting from the intersection of replicate time points were kept using the regular bedtools intersect command. Peaks at time point 0 for WT and OGA-null were then intersected using the bedtools intersect command. Peaks that were less than 20 base pairs were then expanded from their midpoint by 10 base pairs on each side to a peak length of 20 base pairs. This strategy resulted in 1883 peaks and enabled focus on coordinates that consistently contained O-GlcNAz peaks within both WT and OGA-null and each replicate.
O-GlcNAc-seq pilot study: FASTQ sequencing files were aligned to the D. melanogaster (dm3) genome using the snap-aligner single command from SNAP version 1.0beta.18.
O-GlcNAc-seq pilot study: The resulting BAM-format alignment files were sorted, cleared of duplicates and indexed using the SAMtools commands sort, rmdup -s, and index, respectively, using SAMtools version 0.1.19-96b5f2294a
O-GlcNAc-seq pilot study: BDG files were generated from the BAM files using the command bedtools genomecov -ibam -bg command using bedtools v2.17.0.
OGA ChIP-seq: Fastq files were aligned to the dm3 genome the Burrows–Wheeler Aligner version 0.7.5a-r405 algorithm. The resulting BAM-format alignment files were sorted, cleared of duplicates and indexed using the SAMtools commands sort, rmdup -s, and index, respectively, using SAMtools version 0.1.19-96b5f2294a.
OGA ChIP-seq: BDG files were generated from the BAM files using the command bedtools genomecov -ibam -bg command using bedtools v2.17.0.
OGA ChIP-seq: Peaks were called by calculating the coordinates that contained 5 times the average base pair sequencing coverage within both OGA ChIP-seq experiments (WT and OGA-null). Peaks within the OGA-null experiment were considered false positives and were subtracted from the peaks called in the WT experiment using the bedtools intersect -v command to yield the final OGA ChIP-seq peaks.
Genome_build: dm3, sacCer3 and synthetic DNA probes.
Supplementary_files_format_and_content: Bdg files and bed files containing ChIP-seq and O-GlcNAc-seq loci. Bed files of O-GlcNAc-seq data contain normalized sequencing depth at each O-GlcNAc bearing locus accross time points for WT and OGA-null replicates.
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| Submission date |
Jan 08, 2019 |
| Last update date |
Jan 08, 2020 |
| Contact name |
David Vocadlo |
| E-mail(s) |
[email protected]
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| Organization name |
Simon Fraser University
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| Department |
Chemistry
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| Street address |
8888 University Drive
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| City |
Burnaby |
| State/province |
British Columbia |
| ZIP/Postal code |
V5A 1S6 |
| Country |
Canada |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE124785 |
A chemical genetic method for monitoring genome-wide dynamics of O-GlcNAc turnover on chromatin-associated proteins. |
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| Relations |
| BioSample |
SAMN10713488 |
| SRA |
SRX5226983 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3554044_wt.r2.dm3.sacCer3.gblocks-snap.sorted.rmdup.spike-norm_12.bedGraph.gz |
59.9 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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