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| Status |
Public on Sep 11, 2019 |
| Title |
mm10_J2_KO_Pcl2 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cell and NT2 cell line
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| Organisms |
Homo sapiens; Mus musculus |
| Characteristics |
cell type: Embryonic stem cell and NT2 cell line
genotype: Jarid2 KO
antibody: PCL2
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| Growth protocol |
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 17% 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 .
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
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| 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.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Align reads to reference genome (hg19) using bowite2 and the --very-sensitive-local parameter
Align reads to spike-in genome (mm9 or dm6) using bowtie2 and the --very-sensitive-local parameter
Convert sams to sorted bams using Samtools, and extract headers from both alignments for each of the 2 alignments (reference and spike-in)
Filter read ambiguosly aligned to both genomes using Picards FilterSamReads, mark duplicates using Picards MarkDuplicates and index the filtered, marked bam file with Samtools
The number of spike-in reads was used as a scale factor to generate normalised bigWigs using bamCoverage from the deeptools suite with a bin size of 10
Genome_build: hg19, mm9 & dm6
Supplementary_files_format_and_content: bigwig files of ChIP-Rx spike-in normalised tracks for each sample
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| Submission date |
Jun 27, 2019 |
| Last update date |
Sep 12, 2019 |
| Contact name |
Craig Monger |
| E-mail(s) |
MONGERC@tcd.ie
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| Organization name |
Trinity College Dublin
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| Department |
Smurfit Institute of Genetics
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| Street address |
College Green
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| City |
Dublin 2 |
| State/province |
Ireland |
| ZIP/Postal code |
EIRE |
| Country |
Ireland |
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| Platform ID |
GPL19415 |
| Series (2) |
| GSE127121 |
Variant PRC2.1 and PRC2.2 co-operate to direct H3K27 methylations in ESCs |
| GSE133412 |
PRC2.1 and PRC2.2 synergize to co-ordinate H3K27 tri-methylation. |
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| Relations |
| BioSample |
SAMN12147645 |
| SRA |
SRX6370206 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3908361_mm10_J2_KO_Pcl2.bw |
19.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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