GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM3625689 Query DataSets for GSM3625689
Status Public on Sep 11, 2019
Title WT_H3K27me2
Sample type SRA
Source name Embryonic stem cell and S2 cell line
Organisms Drosophila melanogaster; Mus musculus
Characteristics cell type: Embryonic stem cell and S2 cell line
genotype: Wild type
antibody: H3K27me2
Growth protocol Embryonic stem cells (ESCs) were grown on gelatinised culture dishes in GMEM (sigma) supplemented with 10% ES cell qualified FBS (Millipore), 100 U/mL penicillin (Gibco), 100 U/mL streptomycin (Gibco), 50µM β-mercaptoethanol (sigma), 1:100 Glutamax (Gibco), 1:100 non-essential amino acids (Gibco), 1mM sodium pyruvate (Gibco) and 1:500 homemade leukemia inhibitory factor (LIF). S2 cells were cultured in Schneider media supplemented with 10% Heat inactivated FBS
Extracted molecule genomic DNA
Extraction protocol Embryonic stem cells were washed once with PBS before crosslinking for 10 minutes with PBS containing 1% formaldehyde (Sigma). Crosslinking was quenched with 0.125M Glycine for 5 minutes before two PBS washes. The crosslinked cells were lysed in 6mL of SDS-Lysis buffer (100mM NaCl, 50mM Tris pH8.1, 5mM EDTA pH 8.0, 0.02% NaN3, 0.5% SDS, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF). Chromatin was pelleted by centrifugation at 1200rpm for 5 minutes at room temperature. The supernatant was then discarded, and the chromatin was resuspended in 3mL of ChIP buffer (2:1 dilution of SDS-Lysis buffer: Triton dilution buffer [100mM Tris pH 8.6, 100mM NaCl, 5mM EDTA pH 8.0, 0.02% NaN3, 5% Triton X-100, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF]). Chromatin was sheared to approximately 200bp-600bp fragments by sonication on a Branson Sfx150 Sonifier for a total of 4min at 50% amplitude. Sonicated chromatin was incubated overnight with antibody while rotating at 4°C. Following clarification, the chromatin was incubated for 3 hours with 50µL of protein A or G Dyna beads (ThermoFisher) beads. After incubation, the beads were washed three times in Mixed Micelle Buffer (150mM NaCl, 20mM Tris pH 8.1, 5mM EDTA pH 8.0, 5.2% Sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), twice with Buffer 500 (0.1% Sodium Deoxycholate, 1mM EDTA pH 8.0, 50mM HEPES pH7.5, 1% Triton X-100, 0.02% NaN3), twice with LiCl detergent wash (0.5% Sodium Deoxycholate, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 10mM Tris pH 8.0, 0.02% NaN3) and finally one wash with TE. Immunoprecipitated material was eluted from the beads with Elution buffer (0.1M NaHCO3, 1% SDS) while shaking for 1 hour at 65°C. The supernatant was retained and incubated overnight at 65°C while shaking to reverse the crosslinks. The eluted complexes were then subject to RNase (Thermo Fisher) and Proteinase K (Sigma) treatment prior to DNA clean up (Qiagen Qiaquick PCR Purification Kit). ChIP enrichments were analysed by qPCR using the SYBR Green I detection chemistry (M3003E NEB) on an Applied Biosystems Quant Studio 3 platform.
Following the ChIP experiment, the precipitated DNA was quantified using the Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Q32854). A Total of 2-10 ng of DNA from each ChIP-Rx experiment was used for library preparation using the NEBNext Ultra II DNA Library Kit for Illumina (E7645) and NEBNext Multiplex Oligos for Illumina (Set#1, NEB #7335). Following adaptor ligation, DNA was PCR amplified for 5-9 cycles, depending on amount of input DNA. DNA purification was then performed using NEBNext Sample Purification Beads (E7767S). The quality of DNA libraries was analysed on a High Senstivity D1000 Screen Tape (Agilent). The resulting libraries were then used for cluster generation and sequencing using an Illumina NextSeq 500 (ID: NB501524), with 75bp read length.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Description ESCs that have been maintained in a pluripotent state through culturing with LIF on gelatinised plates.ChIPs for certain histone modification ChIP samples include a 1.67% spike in of Drosophila S2 chromatin that were cultured according to established protocols. ChIPs for PRC2 components and H2AK119ub1 include a 10% spike in of human NT2 chroamtin that were cultured according to established protocols .
Data processing Reads were alinged to the mouse reference genome (mm10) and the relevant spike-in genome (dm6 for S2 and hg38 for NT2) using bowtie2.
Bigwig files were generated at a resolution of 10bp using bamCoverage. Tracks were normalised using spike-in derived normalisation factors.
Genome_build: mm10
Submission date Feb 25, 2019
Last update date Sep 12, 2019
Contact name Darren John Fitzpatrick
Organization name Trinity College Dublin
Department Smurfit Institute of Genetics
Street address College Green
City Dublin 2
State/province Ireland
ZIP/Postal code Eire
Country Ireland
Platform ID GPL25537
Series (2)
GSE127117 Variant PRC2.1 and PRC2.2 co-operate to direct H3K27 methylations in ESCs [ChIP-seq]
GSE127121 Variant PRC2.1 and PRC2.2 co-operate to direct H3K27 methylations in ESCs
BioSample SAMN11013734
SRA SRX5426114

Supplementary file Size Download File type/resource 44.9 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap