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| Status |
Public on Jan 04, 2021 |
| Title |
Rtel1f/f Ad-GFP MOCK Input biological replicate 1 |
| Sample type |
SRA |
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| Source name |
Mouse Embryonic Fibroblasts
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| Organism |
Mus musculus |
| Characteristics |
cell type: Mouse Embryonic Fibroblasts
cell line: Rtel1F/F;WT RNH1-GFP MEFs
treatment: Ad-GFP infected;mock treated
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| Treatment protocol |
Rtel1F/F;WT RNH1-GFP MEFs were infected with GFP or Cre-GFP adenovirus, after 48 h doxycycline (2 μg/ml) was added and cells were collected after 48 h
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| Growth protocol |
Rtel1F/F;WT RNH1-GFP MEFs were grown in DMEM supplemented with 10% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x106 cells were collected, washed in PBS and resuspended in 1.6 ml of TRIS-EDTA buffer pH8.0. Cells were lysed by addition 50 μl SDS 20% and 5 μl ProteinaseK 20 mg/ml (Roche), mix gently and incubated overnight at 37°C. DNA was extracted with phenol/chloroform in phase lock tubes, precipitated with EtOH/sodium acetate, washed three times with 70% EtOH, and resuspended in TE. DNA was digested with EcoRI, HindIII, BsrGI, SspI and XbaI (NEB) restriction enzymes overnight at 37°C and DNA was isolated as described above and 4.4 μg of digested DNA was diluted in 500 μl TE buffer pH8.0 aliquots. For DRIP-seq, three IPs with the S9.6 antibody per condition were performed in parallel to obtain enough material for library construction. For each aliquot of digested DNA, 50 μl was kept as input and 50 μl of 10x binding buffer (100 mM NPO4 pH7.0, 1.4 M NaCl, 0.5% Triton X-100) and 10 μl of S9.6 antibody was added to the rest and incubated overnight at 37°C. Protein A Dynabeads (Roche 10002D) were added for 2 h. Bound beads were washed 3 times in binding buffer 1x and elution was performed in elution buffer (50 mM Tris pH 8, 10 mM EDTA, 0.5% SDS, Proteinase K) for 45 min at 55°C. DNA was purified as described and resuspended in 20 μl 10 mM TrisHCl, pH 8.0. Three IPs per condition were pooled and 5 μl was withdrawed and efficiency of IP was assessed by QPCR. Once quality of S9.6 IP was verified, pooled IPs were treated with RNaseA for 1 h at 37°C and sonicated using a Covaris sonicator (11 cycles of 15 sec ON / 90 sec OFF)
Libraries were constructed using the NEB NEBNext® Ultra™ II DNA Library Prep Kit following the manufacturer’s instructions
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
DRIP-seq
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| Data processing |
Library strategy: DRIP-seq
The nf-core/chipseq pipeline (version 1.2.1; Ewels et al., 2020; https://doi.org/10.5281/zenodo.3966161) written in the Nextflow domain specific language (version 19.10.0; Di Tommaso et al., 2017) was used to perform the primary analysis of the samples in conjunction with Singularity (version 2.6.0; Kurtzer et al., 2017).
The command used was " nextflow run nf-core/chipseq --input design.csv --genome mm10 --gtf refseq_genes.gtf --single_end -profile crick -r 1.2.1".
All data was processed relative to the mouse UCSC mm10 genome (Karolchik et al., 2004). Gene annotation files in GTF format were originally downloaded from UCSC on February 19th 2016.
For a condensed overview of the pipeline, the bioinformatics tools used in each step and an extensive list of citations please see the pipeline homepage (https://github.com/nf-core/chipseq).
Genome_build: mm10
Supplementary_files_format_and_content: Normalised genome-wide bigWig coverage files generated from the nf-core/chipseq pipeline command defined above. Normalisation factors were generated by scaling the number of mapped reads to 1 million.
Supplementary_files_format_and_content: MACS2 broadPeak files generated using from the nf-core/chipseq pipeline command defined above.
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| Submission date |
Nov 17, 2020 |
| Last update date |
Jan 04, 2021 |
| Contact name |
Simon J Boulton |
| E-mail(s) |
simon.boulton@crick.ac.uk
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| Organization name |
The Francis Crick Institute
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| Lab |
DSB Repair Metabolism Lab
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| Street address |
1 Midland Rd
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| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE161596 |
Deletion of the helicase RTEL1 in RNA:DNA hybrids throughout the genome and how this may be affected by overexpression of the protein WT RNaseH1GFP in MEFs |
| GSE161597 |
RTEL1 regulates G4/R-loops to avert replication-transcription collisions |
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| Relations |
| BioSample |
SAMN16816047 |
| SRA |
SRX9519252 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4910685_INPUT_GFP_MOCK_R1.bigWig |
363.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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