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| Status |
Public on Feb 27, 2019 |
| Title |
V316C2: GFP-NLS-cGAS1_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Dendritic cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Monocyte-derived dendritic cells
donor: #1
construct: pTRIP-SFFV-EGFP-NLS-FLAG cGAS E225A/D227A
chip antibody: Chromotek GFP-Trap®_MA (cat#gtma-20)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Crosslinking and lysis. 10 million cells were cross-linked in medium with 1% formaldehyde for 8 min at RT on a slow shaker, quenched with freshly prepared 0.125M glycine, incubated 5min at RT on a slow shaker, then pelleted at 400g for 5 minutes at 4°C, washed three times with 30ml of ice cold PBS and then incubated for 20 minutes rotating at 4°C in 1mL of RIPA lysis buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0 (Invitrogen), 140mM NaCl, 1% (v/v) Triton X-100 (Euromedex), 0.1% (v/v) SDS and 0.1% sodium deoxycholate (SIGMA)). Nuclei were pelleted at 1350g for 5 minutes at 4°C, washed for 10 minutes rotating with 1ml of a buffer containing 10mM Tris, 200mM NaCl, 1mM EDTA (Invitrogen), 0.5 mM EGTA (Euromedex), pelleted and lysed in buffer containing 0.4% SDS (Euromedex), 10mM EDTA (Invitrogen), 50mM Tris-HCl pH 8.0 for 30min on ice (volume of buffer = 100µl/1.6 million cells). Lysates were sonicated on a Bioruptor Pico (Diagenode) sonication devices (11cycles 30 seconds ON, 30 second OFF) to reach fragments ranging from 150 to 500bp, and then centrifuged at 10,000g for 10 minutes at 4°C to remove debris. Samples were then snap-frozen in liquid nitrogen and stored at -80°C until immunoprecipitation. All buffers contained cOmplete EDTA free Protease inhibitor cocktail (Roche). Immunoprecipitation. Lysates were pre-cleared for 15 minutes rotating using 30µl of Binding Control magnetic agarose beads (Chromotek). Chromatin was diluted four-fold in dilution buffer containing 20mM Tris-HCl pH 8.0, 1% Triton X-100 (Euromedex), 2mM EDTA (Invitrogen), 167mM NaCl. 1% of the diluted lysate was recovered and used as input. For GFP-trap and control beads, chromatin was incubated for 5 hours in the presence of 0.1% BSA (Euromedex) (30µl of beads (GFP-Trap_MA beads (Chromotek) or Control magnetic agarose beads (Chromotek)/600µl per Eppendorf tube of the diluted lysate). Lysates were washed on a 96 well plate magnet with low salt washing buffer (140mM NaCl) (5 times), high salt washing buffer (500mM NaCl) (2 times), high LiCl washing buffer (250mM LiCl) (2 times), TE Buffer (Invitrogen) (1 time). All wash buffers were diluted in RIPA buffer 10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0 (Invitrogen), 140mM NaCl, 1% (v/v) Triton X-100 (Euromedex), 0.1% (v/v) SDS and 0.1% sodium deoxycholate (SIGMA)) and contained cOmplete EDTA free Protease inhibitor cocktail (Roche). DNA purification. DNA was eluted in elution buffer (1% SDS, 50 mM NaHCO3) by shacking 2h at 37°C (100µl of buffer/tube) with 10 μg/mL RNaseA (Thermo Fischer), then 4h with 0.2 μg/ml proteinase K. Beads were concentrated on the magnet and take out eluate. Samples were decrosslinked overnight at 65°C. Inputs were treated like ChIP samples. DNA was purified by phenol/chloroform/isoamyl alcohol (SIGMA) followed by purification on MinElute columns (Qiagen). DNA was eluted in 50µl of H2O and DNA concentration was measured with a Qubit fluorometer (Thermo Fischer).
Traces of high molecular weight fragments were eliminated with SPRIselect beads (Beckman Coulter). Illumina TruSeqChIP library prep kit was used to prepare indexed libraries from IP and Input DNA. Libraries were pooled respecting equimolarity. Sequencing was performed on Illumina MiSeq sequencer in 150 bp paired-end reads (replicate 1 of input and GFP-NLS-cGAS IP), 100 bp single-end reads (replicates 2 and 3 of input, GFP-NLS and GFP-NLS-cGAS IP).
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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 |
Reads for each replicate were mapped separately to the hg38 primary assembly with Bowtie2 v2.2.9 using a seed length of 22bp with at most 1 mismatches (-N 1 -L 22) and keeping the best scoring alignment per read.
Duplicate fragments were identified with MarkDuplicates from picard v1. Only non-duplicate, properly paired reads (same reference, inner-oriented, insert size <= 500bp; only for replicate 1) with mapping quality >= 20 were retained, using samtools v1.3. Alignment files were converted from BAM to BED/BEDPE format using bedtools bamtobed from bedtools v2.27.1.
Genome mappability was computed with gemtools v1.7.1 (--l L --m 0.04 --e 0.04 --max-big-indel-length 15 --min-matched-bases 0.80, where L is the read length). The effective genome size was then defined as m/n , where m is the number of bp with mappability score 1 and n is the genome length.
Peak calling of GFP-NLS-cGAS IP reads on hg38 chromosomes was performed with SICER v1.1 on each replicate separately, using either input or GFP-NLS IP (only for replicate 2 and 3) reads as background (redundancy threshold 1, window size 200bp, FDR 0.05, effective genome size estimated with the above procedure). The gap size was set to 600bp for replicate 1 and to 400bp for replicate 2 and 3. For replicate 1, one read for each pair was used and the fragment size was set to the average insert size computed from the alignment.
Filtered peaks were defined for each replicate i as the peaks supported by more than M ChIP reads, where M is the median across all peaks for replicate i.
The number of reads for each repeat element is computed on the annotated hg38 genome with a in-house C++ program and the cGAS enrichment over either input or GFP is retrieved for each replicate separately.
All repeat elements in the genome are ranked according to the cGAS read enrichment value and grouped into n ranking bins. The number of elements falling into each bin is computed for each repeat class R.
Genome_build: hg38
Supplementary_files_format_and_content: Bed files generated with SICER containing the peaks, which are subsequently filtered by the number of ChIP reads.
Supplementary_files_format_and_content: Tabular files containing the count and the normalized count (cpm) of the reads mapped hg38 to each annotated repeat locus
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| Submission date |
Jan 22, 2019 |
| Last update date |
Feb 24, 2020 |
| Contact name |
Nicolas Manel |
| E-mail(s) |
nicolas.manel@curie.fr
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| Organization name |
Institut Curie
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| Department |
INSERM U932
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| Street address |
12 rue Lhomond
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| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE125431 |
Nuclear GFP ChIP-seq from human Monocyte Derived Dendritic Cells transduced with GFP-NLS-cGAS or GFP-NLS |
| GSE125475 |
The N-terminal domain of cGAS determines preferential association with centromeric DNA and innate immune activation in the nucleus |
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| Relations |
| BioSample |
SAMN10787853 |
| SRA |
SRX5276081 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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