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| Status |
Public on Mar 22, 2023 |
| Title |
5_NR4A2 |
| Sample type |
SRA |
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| Source name |
CD1c+ DCs
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD1c+ dendritic cell
tissue: Periperal blood
replicate: 3
treatement: unstimulated
chip antibody: anti-NR4A2 (NB110-40415, Novusbio)
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| Treatment protocol |
Cells were either left untreated or treated with TLR7/8 agonist R848 (Resiquimod, 100ng/mL, Invivogen) overnight.
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| Growth protocol |
CD1c+ cDCs were isolated from PBMCs obtained from healthy donor buffy coats using the MACS human CD1c dendritic cell isolation kit. 1.5×106 freshly isolated cDCs were cultured overnight in RPMI 1640 medium with GlutaMAX™ (Life Technologies), supplemented with 10% heat inactived fetal bovine serum (Biowest) and 1% penicillin streptomycin (Life Technologies).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
After culture, cDCs were washed with PBS and crosslinked using the truChIP® Ultra-Low Chromatin Shearing Kit (Covaris), according to the manufacturer’s instructions. Chromatin shearing was performed using AFA Fiber Pre-Slit Snap-Cap microTUBEs (Covaris) by sonication (peak incident power: 105, duty factor: 2%, cycles per burst: 200, treatment time: 12 minutes) on the Covaris S220 focused ultrasonicator (Woburn, MA, USA). After shearing, the chromatin was transferred into pre-chilled microcentrifuge tubes and centrifuged at 10.000 x g, 4˚C for 5 minutes to pellet insoluble material, and the chromatin was stored at -20˚C for downstream processing. Chromatin immunoprecipitation was performed with 3µg anti-NR4A1 (NB100-56745, Novusbio), anti-NR4A2 (NB110-40415, Novusbio) or anti-NR4A3 (NLS2341, Novusbio) antibodies, using the low cell ChIP-seq kit (Active Motif), according to the manufacturer’s instructions. For all conditions, 10% of the input chromatin was removed prior to addition of the antibodies and used to normalize the amount of immunoprecipitated DNA (input control). Following immunoprecipitation, ChIP DNA was de-crosslinked in the presence of NaCl and Proteinase K (Active Motif) in a thermocycler at 65°C overnight, and DNA was extracted by penol:chloroform:isoamylalcohol precipitation and dissolved in Low-EDTA TE buffer (Active Motif).
ChIP-seq libraries were generated by GenomeScan (Leiden, the Netherlands) with the NEBNext® Ultra II DNA Library Prep kit (Illumina), and were sequenced using Illumina NovaSeq 6000 generating ~20 million 150bp paired ended reads for each sample.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
H2GNHDSX2_104400-001-019_AGGAACAC-GACATTCC
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| Data processing |
Quality check of the raw sequencing reads was performed using the FastQC tool.
Sequencing reads were mapped against the reference genome GRCh38.p13 built from the human genome (NCBI) using bowtie2
Peaks were called using MACS2, by comparing the IP samples to their matched input samples in paired end mode (BAMPE).
After calling, peaks from ENCODE blacklist regions and X- and Y-chromosome peaks were filtered out to reduce noise and exclude sex-specific peaks.
Peaks were annotated to the nearest genes using the ChIPseeker Bioconductor/R package. Peaks were considered to be associated to a gene when they were annotated within a 10kb range (up- or downstream) to the gene transcription start site (TSS).
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: peak text files and bed files
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| Submission date |
Oct 20, 2021 |
| Last update date |
Mar 22, 2023 |
| Contact name |
Aridaman Pandit |
| Organization name |
University Medical Center Utrecht
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| Street address |
Heidelberglaan 100
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| City |
Utrecht |
| ZIP/Postal code |
3584 CX |
| Country |
Netherlands |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE186197 |
Nuclear receptor subfamily 4A signaling as a key disease pathway of CD1c+ dendritic cell dysregulation in systemic sclerosis [ChIP-seq] |
| GSE186199 |
Nuclear receptor subfamily 4A signaling as a key disease pathway of CD1c+ dendritic cell dysregulation in systemic sclerosis |
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| Relations |
| BioSample |
SAMN22420518 |
| SRA |
SRX12698905 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5640217_104400-001-019_ChIP_summits.bed.gz |
170.0 Kb |
(ftp)(http) |
BED |
| GSM5640217_5_NR4A2.txt.gz |
48.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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