|
| Status |
Public on Feb 01, 2021 |
| Title |
WDR82-ChIPseq_LPS_45min_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Macrophages
|
| Organism |
Mus musculus |
| Characteristics |
cell type: RAW 264.7 Macrophages
genotype: wild type
strain: BALB/c
chip antibody: anti-WDR82 (Cell Signaling Technologies, cat.no. 99751, clone D2I3B)
|
| Treatment protocol |
Cells were treated for 45 minutes with LPS (10 ng/ml) before harvesting.
|
| Growth protocol |
RAW 264.7 mouse macrophage cell line was purchased from the American Type Culture Collection (ATCC). Cells were grown in DMEM containing 10% South American FBS, 1% L-Glutamax solution and 1% Pen/Strep solution.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Ca. 200 mill. cells were pelleted and resuspended in PBS and fixed first with disuccinimidyl glutarate at 2mM final concentration for for 45 min at room temperature, followed by 1% formaldehyde fixation for 10 min. Nuclear lysates were prepared, sonicated to reach an average fragment length of 300 bp, whereafter protein-DNA complexes were immunoprecipitated with specific antibodies.
DNA libraries were prepared for sequencing on the NextSeq as previously described (Ostuni, 2013).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Chromatin IP against WDR82
|
| Data processing |
Single end reads (76bp) were trimmed and clipped for quality control with Trimmomatic v0.27 (PMID:24695404). Read quality was then checked for each sample using FastQC v0.10.1 (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). High-quality reads were mapped using Bowtie2 v 2.2.6 (PMID: 19261174). We used default parameters with the options --very-sensitive, --no-unal and with the pre-built bowtie2 index. Only uniquely mapping reads were retained.
Peak calling was performed using SICER v2 (PMID: 24743992). We identified significantly enriched clusters using a redundancy threshold of 1, a window size of 200 bp, a gap size of 600 bp and a False Discovery Rate (FDR) cut-off <0.01 were used. Fragment size was set to 150nt and the effective genome fraction to 0.80. The ChIP was compared to input DNA derived from RAW264.7 mouse macrophages. Regions that overlapped with the blacklists of the ENCODE and modENCODE consortia (http://www.broadinstitute.org/~anshul/projects/mouse/blacklist/mm9-blacklist.bed.gz) were filtered out.
Tracks (*.bigWig files) were generated using bamCoverage from deepTools v.3.1.3 (PMID:27079975)
Genome_build: mm10 (GENCODE) https://www.gencodegenes.org/mouse/release_M24.html
Supplementary_files_format_and_content: bigWig
|
| |
|
| Submission date |
Jun 20, 2019 |
| Last update date |
Feb 02, 2021 |
| Contact name |
Viviana Piccolo |
| E-mail(s) |
viviana.piccolo@ieo.it
|
| Organization name |
European Institute of Oncology (IEO)
|
| Department |
Department of Experimental Oncology
|
| Lab |
Gioacchino Natoli Lab
|
| Street address |
Via Adamello 16
|
| City |
Milano |
| State/province |
Milano |
| ZIP/Postal code |
20139 |
| Country |
Italy |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE133103 |
A first exon termination checkpoint that preferentially suppresses extragenic transcription [ChIP-seq Raw W ZC] |
| GSE133109 |
A first exon termination checkpoint that preferentially suppresses extragenic transcription |
|
| Relations |
| BioSample |
SAMN12100518 |
| SRA |
SRX6099383 |
