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Sample GSM2238594 Query DataSets for GSM2238594
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
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.
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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