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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 19, 2021 |
| Title |
K27me3_C2plusTplusE2_Daniel |
| Sample type |
SRA |
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| Source name |
Immortalized mouse embryonic fibroblast line A, Ezh2 -/- , retroviral pMSCV-Ezh2
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| Organism |
Mus musculus |
| Characteristics |
cell type: c-myc-immortalized mouse embryonic fibroblasts
genotype: Ezh2 -/- , retroviral pMSCV-Ezh2
chip antibody: H3K27me3 (Cell Signaling Technology, clone C36B11), catalog# 9733
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| Growth protocol |
MEF cells were grown in DMEM supplemented with 10% FCS, 100 mM nonessential amino acids, 2 mM L-glutamine.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq, lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with specific antibodies. For CUT&RUN permeabilized cells were incubated with specific antibodies followed by incubation with pA-MNAse. Digested DNA fragments were subsequently recovered in the supernatant.
ChIP-seq libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Alignment to mouse and Drosophila genomes (CUT&RUN, NPC samples) or to mouse genome (Traditional ChIP, others) (Bowtie 2.2.9)
Removal of PCR duplicates (Picard Tools MarkDuplicates 1.97)
Filtering of reads to exclude common artifact regions (https://github.com/Boyle-Lab/Blacklist/tree/master/lists) and to retain only autosomal reads with MAPQ>10
Calculation of scale factors from Drosophila reads counted in 10kb bins using the median ration method (CUT&RUN, NPC samples) (DESeq2 estimateSizeFactors 1.22.2)
Calculation of RPKM values per 50bp bin for mouse reads, multiplication by scale factors and conversion to bigWig format (CUT&RUN, NPC samples) (DeepTools bamCoverage)
Peak calling on combined replicates using MACS2 (2.1.1.20160309) with parameters -f BAMPE --gsize mm --broad --broad-cutoff 0.1 --bdg
Genome_build: mm10, dm6
Supplementary_files_format_and_content: .bw files contain alignments (reads per kilobase per million within bins)
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| Submission date |
Mar 26, 2020 |
| Last update date |
Aug 19, 2021 |
| Contact name |
Daniel Holoch |
| E-mail(s) |
daniel.holoch@curie.fr
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| Phone |
0156246553
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| Organization name |
Institut Curie, Centre de Recherche
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| Department |
Genetics and Developmental Biology Unit
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| Lab |
Raphaël Margueron
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| Street address |
26 rue d'Ulm
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| City |
Paris |
| State/province |
Cedex 05 |
| ZIP/Postal code |
75248 |
| Country |
France |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE147567 |
A cis-acting mechanism mediates trancriptional memory at Polycomb target genes in mammals [ChIP-Seq] |
| GSE147568 |
A cis-acting mechanism mediates trancriptional memory at Polycomb target genes in mammals |
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| Relations |
| BioSample |
SAMN14452964 |
| SRA |
SRX8006746 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4434068_K27me3_C2plusTplusE2_Daniel_rpkm.bw |
104.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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