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Sample GSM2238595 Query DataSets for GSM2238595
Status Public on Jul 18, 2017
Title Pho ChIP-seq in S2 cells
Sample type SRA
Source name s2-cells-pho
Organism Drosophila melanogaster
Characteristics tissue: Schneider's Drosophila Line 2 (SL2) cells
cell line: SL2
passages: 10 to 12
chip antibody/selection: Pho antibody
Biomaterial provider ATCC [D. Mel. (2), SL2] (ATCC® CRL-1963™)
Treatment protocol Cells were not treated.
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). 120 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 1:100 anti-Pho antibody (kindly provided by Judith Kassis1) for 12 hours at 4°C. 100 µL protein A/G agarose beads (Calbiochem), previously blocked with 1 mg/mL BSA, was added to each chromatin at 4°C overnight. 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).
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 SAMN05413710
SRA SRX1958586

Supplementary file Size Download File type/resource
GSM2238595_s2-cells-pho.bedgraph.gz 57.6 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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