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| Status |
Public on Jul 18, 2017 |
| Title |
GlcNAc ChIP-seq on S2 cells using WGA purification |
| Sample type |
SRA |
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| Source name |
s2-cells-wga
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Schneider's Drosophila Line 2 (SL2) cells
cell line: SL2
passages: 10 to 12
chip antibody/selection: Purification with WGA resin
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| Biomaterial provider |
ATCC [D. Mel. (2), SL2] (ATCC® CRL-1963™)
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| Treatment protocol |
Cells were not treated.
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| Growth protocol |
Drosophila melanogaster Schneider S2 cells were cultured in Sf-900 II SFM medium (Invitrogen) supplemented 100 U/ml Penicillin and 100 mg/ml Streptomycin at 25°C. Cells were passaged at 1:4 ratio every two days to keep logarithmic growth.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
~2x107 cells were fixed for 10 min at room temperature by gently mixing in 10 mL crosslinking solution (1% formaldehyde, 50 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), crosslinking was quenched by adding 2M glycine to a final concentration of 240 mM. The cells were sequentially collected by centrifugation at 700 x g for 10 min and washed trice with 10 mL ice-cold PBS. These cell pellets were sonicated in parallel to obtain chromatin fragments of ~200 to 700 bp, each in 4 mL sonication buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, Roche protease inhibitor cocktail) with a Sonic Dismembrator Model 500 (Fisher Scientific, 10 x 30 seconds on / 45 seconds off cycles, 50% power settings). After sonication, debris was removed by centrifugation at 4°C for 10 minutes at 13,000 rpm, and equal amount of 6M urea was added to the solution then incubated for 10 minutes at 4°C on a rotating wheel. The soluble chromatin was dialysed using a membrane with a molecular weight cut-off of 3.5 kDa, at 4°C against 2 L dialysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 4% glycerol) overnight. Insoluble debris was removed by quick centrifugation (7,500 rpm, 2 min).20 μg of extract were incubated in a final volume of 1 mL IP buffer (15 mM Hepes pH 7.9 / 200 mM KCl / 1.5 mM MgCl2 / 0.2 mM EDTA pH 8 / 0.25% NP-40 / 20% glycerol / 0.3 mM DTT / 1x "Complete" protease inhibitor cocktail / 1 mM PMSF) with 100 μl of a 50% slurry of washed succinylated WGA-agarose resin (Vector Labs) for 12 hours at 4°C. Note: The length of these washes can range from 10 min to overnight. An overnight wash can significantly reduce background signal. The length of washing was optimized by examination of the background present in the control and optimizing washing times to reduce this background signal. Succinylated WGA has high affinity for GlcNAc, and a reduced affinity for sialic acids. Beads were washed with IP buffer containing 0.5 mM DTT and 0.4% NP-40, followed by a 1 hour incubation with 1 M GlcNAc (Galab) on ice to elute resin-bound proteins
The libraries were prepared according to the manufacturer's instructions using the NEBNext kit (E6200). Briefly, DNA was fragmented by sonication to a maximum of 300 bp. Next the ends of the fragments were repaired with a combination of fill-in reactions and exonuclease activity to produce blunt ends that were then tailed with an A-base. Illumina-specific Adaptors were ligated followed by removal of unligated adaptors using AMpure XP beads (Beckman Coulter). Finally a PCR with 12-15 cycles for both kits was performed to enrich final adaptor-ligated fragments. Quality and quantity were assessed on an Agilent Bioanalyzer Chip DNA 7500 or High Sensitivity. For all libraries, sequencing was performed on the MiSeq platform, using v2, 300-cycle reagent kits (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Data processing |
ChIP-seq reads were aligned to the mm9 genome assembly using bwa-mem or bwa-bwasw algorithm version 0.7.5a-r405.
Bam-format alignment files were generated with Samtools version 0.1.19-96b5f2294a.
Multiple runs were merged with Samtools.
Duplicate read-pairs were removed with Samtools.
BDG files generated with macs version1.4
Genome_build: dm3
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| Submission date |
Jul 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
David Vocadlo |
| E-mail(s) |
dvocadlo@sfu.ca
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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 |
GPL16479 |
| Series (1) |
| GSE84502 |
Genome-wide maps of GlcNAc bound proteins and Pho ChIP-seq in Drosophila S2 cells and pupae. |
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| Relations |
| BioSample |
SAMN05413711 |
| SRA |
SRX1958587 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2238596_s2-cells-wga.bedgraph.gz |
48.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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