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| Status |
Public on Mar 06, 2017 |
| Title |
PMLKO_H33_HA |
| Sample type |
SRA |
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| Source name |
Mouse embryonic fibroblasts, PML knock-out
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: MEF
genotype/variation: PML knock-out
passages: Passage 10-15
state/treatment: Proliferating, untreated
chip antibody: anti-HA (Abcam, catalog# ab-9110, lot# GR235874-8)
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| Treatment protocol |
Cells were cultured under proliferating conditions until confluency. When confluent, cells were harvested by trypsinization and crosslinked for ChIP (H3K9me3, H3K27me3, PML). Cells were also collected and transfected using a nucleofector (Amaxa) with relevant plasmids (H3.3-Flag or H3.3-HA), with or without transfection with relevant siRNAs as described in the acompanying paper. After transfection(s), cells were reseeded and cultured as above prior to harvest and crosslinking for ChIP. For RNA-seq experiments, only native cells (non-transfected) were used.
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| Growth protocol |
MEFs isolated from wild-type and Pml-null mice (C57BL/6 background) were cultured in T175 flasks in DMEM/F12 supplemented with 10% fetal calf serum and 1% penicillin streptomycin. Cells were cultured to confluency prior to ChIP.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq: Cells (10e7 cells/ChIP) were harvested and fixed with 1% formaldehyde for 10 min before quenching with 125 mM glycine. Cells were lysed for 30 min at 4C on a rotator in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% deoxycholate, 1 mM PMSF, protease inhibitor cocktail) adjusted to 1% SDS, and sonicated 3 times 15 min in a Bioruptor® (Diagenode). After sedimentation at 10,000 g for 10 min, the supernatant was collected and diluted 10x in RIPA buffer without SDS to constitute input chromatin. Chromatin was incubated overnight at 4oC on a rotator with antibodies coupled to magnetic Dynabeads Protein G. ChIP samples were washed 3 times in ice-cold RIPA buffer. Crosslinks were reversed and DNA eluted for 6 h at 37C in 50 mM NaCl, 20 mM Tris-HCl pH 7.5, 5 mM EDTA, 1% SDS, 0.5 µg/ml RNase A, and 2 µg/ml Proteinase K added twice. DNA was purified and dissolved in H2O.For RNA-seq: 10e6 cells were cultured. Total RNA was isolated using the Ambion TRIzol® Reagent RNA extraction kit (Life Technologies), quality checked using a BioAnalyzer 2100 and processed for library preparation.
Sequencing library was prepared according to the Illumina protocol, and sequenced on an Illumina HiSeq2500 and NextSeq500 at the Norwegian Sequencing Center.
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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 |
For ChIP: reads aligned to reference using bowtie version 1.0.0 using parameters –best
For ChIP: bigWig files were generated by getting a ratio of IP over Input in 1 kb bins (H3, H3K9me3, PML) and consist of raw counts (H3.3 and H3K27me3)
For ChIP: peaks called using Enriched Domain Detector (H3, PML) and RSEG (H3.3, H3K27me3, H3K9me3)
For RNA: reads aligned with tophat v2.0.8b with parameter –-b2-very-sensitive and -G
For RNA: transcript abundance estimated with cufflinks v2.2.1 with parameter -–GTF -b and -u
For RNA: reference gtf with cuffmerge v2.2.1 with parameters -g, -s
For RNA: diffrential expression analysis with cuffdiff v2.2.1 with parameters -b, -u
For RNA: csv file created from cuffdiff output file genes.fpkm_tracking
Genome_build: mm10
Supplementary_files_format_and_content: [ChIP-Seq] bigWig files for viewing in Genome Browser; bed files with columns: Chromosome, Start, End, Score; or columns: Chromosome, Start, End, Name/Blank, Score; or columns: Chromosome, Start, End, Name/Blank, AverageCount, DomainScore, Strand
Supplementary_files_format_and_content: [RNA-Seq] .cvs file reports FPKMs
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| Submission date |
Jul 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Philippe Collas |
| Organization name |
University of Oslo
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| Department |
Institute of Basic Medical Sciences
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| Street address |
PO Box 1112 Blindern
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| City |
Oslo |
| ZIP/Postal code |
0317 |
| Country |
Norway |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE66364 |
PML protein organizes heterochromatin domains where it regulates histone H3.3 deposition by ATRX/DAXX |
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| Relations |
| BioSample |
SAMN05416366 |
| SRA |
SRX1960056 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2241939_PMLKO_H33_HA.bw |
318.6 Mb |
(ftp)(http) |
BW |
| GSM2241939_PMLKO_H33_HA_peaks.bed.gz |
28.8 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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