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Status |
Public on Mar 02, 2017 |
Title |
ChIP-Seq H3K27ac_IFNg_4h |
Sample type |
SRA |
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Source name |
Primary bone marrow-derived macrophages (BMDM)
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 cell type: BMDM cells (7th day of differentiation)
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Treatment protocol |
Macrophages were subjected to different treatment (see individual samples for details)
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Growth protocol |
Bone marrow cells isolated from C57BL/6 mice were plated in 10 cm plates in 5ml of BM-medium (high glucose DMEM supplemented with 20% low endotoxin fetal bovine serum, 30% L929-conditioned medium, 1% glutamine, 1%, Pen/Strep, 0.5% Sodium Pyruvate, 0.1% β-mercaptoethanol). Cultures were fed with 2.5 ml of fresh medium every two days.
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Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP lysates were generated from 2x10^8 cells. Cells were fixed in 1% formaldehyde for 10min. Lysate was immunoprecipitated with 10ug of antibody. Antibodies were pre-bound overnight to 100ul of G protein-coupled paramagnetic beads in PBS/BSA 0.5%. Beads were then added to lysates (the preclearing step was omitted) and incubation was let to proceed overnight. Beads were washed 6 times in a modified RIPA buffer (50mM Hepes pH 7.6, 500mM LiCl, 1mM EDTA, 1% NP-40, 0.7% Na-deoxycholate) and once in TE containing 50mM NaCl. DNA was eluted in TE containing 2% SDS and crosslinks reversed by incubation overnight at 65ºC. DNA was then purified by Qiaquick columns (Qiagen) and quantified using PicoGreen (Invitrogen). ChIP DNA was prepared for HiSeq 2000 Illumina sequencing using a standard protocol consisting in blunting, addition of dA overhangs, ligation of Illumina adapters, selection on gel and PCR with index primers. A mixture of T4 DNA polymerase, DNA polymerase I and T4 kinase was used according to manufacturer’s instruction. Library preparation is carried out on SPRIworks Fragment Library System.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer II |
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Description |
antibody: Anti-H3K27ac (Abcam # ab4729) treatment: IFNg (100 ng/ml) for 4hrs Chromatin IP against H3K27ac
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Data processing |
Reads quality filtered according to the Illumina pipeline Reads were mapped to the UCSC mm10 reference genome using Bowtie2 v.2.2.6 (PMID: 22388286) with "--very-sensitive" parameter. Subsequently, only uniquely mapping reads were considered in the analysis. Peaks were called using using SICER v1.1 (PMID: 24743992) using a redundancy threshold of 1, a window size of 200 bp, a gap size of 600 bp and a False Discovery Rate (FDR) cutoff of 1 × 10−3. Fragment size was set to 150 and the effective genome fraction to 0.80. Genome_build: mm10 (Genome Reference Consortium GRCm38 Supplementary_files_format_and_content: Summary file of significant islands with requirement of FDR=0.01 detected with SICER. It is a bed format with the end position included : chrom, start, end, ChIP_island_read_count, CONTROL_island_read_count, p_value, fold_change, FDR_threshold
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Submission date |
Jul 18, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Viviana Piccolo |
E-mail(s) |
viviana.piccolo@ieo.it
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Organization name |
European Institute of Oncology (IEO)
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Department |
Department of Experimental Oncology
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Lab |
Gioacchino Natoli Lab
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Street address |
Via Adamello 16
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City |
Milano |
State/province |
Milano |
ZIP/Postal code |
20139 |
Country |
Italy |
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Platform ID |
GPL9250 |
Series (2) |
GSE84519 |
Opposing macrophage-polarization programs show extensive epigenomic and transcriptional cross-talk [ChIP_broadly] |
GSE84520 |
Opposing macrophage-polarization programs show extensive epigenomic and transcriptional cross-talk |
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Relations |
BioSample |
SAMN05414030 |
SRA |
SRX1958828 |
Supplementary file |
Size |
Download |
File type/resource |
GSM2241208_H3K27ac_IFNg_4h_R1_bw2_mm10_unique.mapped-W200-G600-islands-summary-FDR.01.bed.gz |
1.0 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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