 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 18, 2017 |
| Title |
GlcNAc ChIP-seq on GalNAz fed S2 cells |
| Sample type |
SRA |
| |
|
| Source name |
s2-cells-galnaz
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Schneider's Drosophila Line 2 (SL2) cells
cell line: SL2
passages: 10 to 12
chip antibody/selection: GalNAz feeding, Staudinger ligation, and streptavidin purification
|
| Biomaterial provider |
ATCC [D. Mel. (2), SL2] (ATCC® CRL-1963™)
|
| Treatment protocol |
For labeling, media was aspirated and the cells were washed with PBS. DMSO stocks of Ac4GlcNAz was added to achieve the final treatment conditions and incubated for 16-24 h, with vehicle-only controls always included. ~3.3x106 S2 cells were used as a unit of cells for each experiment. This amount of cells yeilded more that enough DNA and protein for downstream analysis and ensured a large biological sample size.
|
| 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.
|
| 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). Before each enrichment of O-GlcNAz modified nuclear proteins, 500 µL solution (1% SDS/PBS) consisting of 800-1,000 µg nuclear protein was mixed with 50 µL Streptavidin-agarose slurry (Sigma), incubated for 1 h at 4°C for preclearing. The supernatant was transferred to a new tube and reacted with 200 µM Biotin-azo-phosphine for overnight at room temperature. Unreacted probe was removed by chloroform/methanol precipitation. Air-dried protein pellets were resuspended in 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0) and enriched by Streptavidin-agarose slurry (Sigma) for a couple of hours at 4 °C with gentle rocking. The beads were sequentially washed trice with 5-10 volumes of 6 M urea/2 M thiourea/10 mM HEPES (pH 8.0), PBS, and 1% SDS/PBS. Centrifugation of the beads between washing steps was carried out (7,500 rpm, 2 min). Bound proteins were cleaved from the beads by treating with one beads volume of elution buffer (100 mM Na2S2O4 in 1% SDS/PBS) for 30 min trice. Collect and combine the eluants. Removal of the majority of Na2S2O4 from the eluants was achieved by precipitating them with chloroform/methanol method; 10% of each eluted sample was analyzed by Immunoblot using Odyssey (LI-COR Biosciences).
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).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
| |
|
| 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
|
| |
|
| Submission date |
Jul 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
David Vocadlo |
| E-mail(s) |
dvocadlo@sfu.ca
|
| Organization name |
Simon Fraser University
|
| Department |
Chemistry
|
| Street address |
8888 University Drive
|
| City |
Burnaby |
| State/province |
British Columbia |
| ZIP/Postal code |
V5A 1S6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16479 |
| Series (1) |
| GSE84502 |
Genome-wide maps of GlcNAc bound proteins and Pho ChIP-seq in Drosophila S2 cells and pupae. |
|
| Relations |
| BioSample |
SAMN05413709 |
| SRA |
SRX1958585 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2238594_s2-cells-galnaz.bedgraph.gz |
64.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
