|
|
GEO help: Mouse over screen elements for information. |
|
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 Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
|
|
|
|
|