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Sample GSM3625692 Query DataSets for GSM3625692
Status Public on Sep 11, 2019
Title TKO_H3K27me1
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: Pcl1-3 TKO
antibody: H3K27me1
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
E-mail(s) fitzpadj@tcd.ie
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
Relations
BioSample SAMN11013731
SRA SRX5426117

Supplementary file Size Download File type/resource
GSM3625692_mm10_KO_H3K27me1.bw 99.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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