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| Status |
Public on Dec 31, 2023 |
| Title |
DRIP, ALS, K039 |
| Sample type |
SRA |
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| Source name |
blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: blood
patient code: K039
Sex: female
age (at_sampling): 52
age (at_als_onset): N/A
disease: none
symptoms: N/A
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA extraction from whole blood. We prepared aliquots of 1 ml frozen whole blood sample from ALS and control patients and extracted 200 μL of it using the Macherey-Nagel™ NucleoSpin™ Tissue Kit according to the manufacturer's protocol.
DNA-RNA Immunoprecipitation (DRIP) sequencing was started by blocking and coating magnetic beads (Dynal protein G). 2x50 μL of protein G beads were washed with 3x 1 ml of 5 mg/ml PBS/1mM EDTA/1% BSA solution using a MagnaRack. To the beads, we added 500 µl of 5 mg/ml PBS/1mM EDTA/1% BSA solution and 25 μg of the RNA-DNA hybrid-specific S9.6 monoclonal antibody. The mixture was incubated for 4 hours with rotation at 4°C in a cold room and then washed with 1 ml of PBS/1mM EDTA/1% BSA twice. For immunoprecipitation, we diluted each sample to 700 µl with ChIP lysis Buffer (50 mM Hepes/KOH pH 7,5, 0,14 M NaCl, 5 mM EDTA, 1% Triton X-100, 0,1 % Na-Deoxycholate). We mixed 200 μL of the sample with 500 μL of ChIP lysis Buffer and added 2 x 700 µl of this mixture to 50 µl of S9.6-coated beads. After incubating overnight at 4°C with rotation, we performed a series of washing steps using different buffers as described (Halász et al. 2017, PMID: 28341774). Finally, the DNA was eluted from the beads by adding 100 µl of IP Elution buffer (50 mM Tris/HCl pH 8.0, 10 mM EDTA, 1% SDS), vortexing every 2 minutes, and incubating in a thermomixer for 15 minutes at 65°C. We then used a MagnaRack to separate the supernatant and pipetted it into a new low-binding tube. To further purify the DNA, we conducted a PCR clean-up using the NTB M&N kit, eluting the DNA in 3x 100 μL of H2O (pH >7). Before NGS library preparation, we performed further fragmentation through sonication in a 1.5 ml low-bind tube (300 μl sample) using a Bioruptor (Diagenode) with 2 x 4 cycles of 30 seconds ON/OFF at LOW settings. Finally, we concentrated the samples to 30 μL using a speed vacuum concentrator and measured the concentration using the Qubit dsDNA HS Assay Kit. NGS library preparation and sequencing was performed as described (Karányi et al. 2022, PMID: 36030240; Feró et al. 2023, PMID: 37286661) using the Illumina TruSeq ChIP Sample Preparation protocol.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequencing was performed using an Illumina HiSeq 2500 instrument (at the EMBL Genomics Core Facility, Heidelberg) in the 2x125 paired-end sequencing mode.
DRIP-seq reads were aligned to the GRCh38 reference sequence using bowtie2 v2.3.5.1 (default parameters).
Alignments were filtered using samtools v1.10 keeping only primary alignments of properly paired reads that had >30 mapping quality score. PCR and optical duplicates were also filtered out. (Parameters: samtools view -q30 -f3 -F3840.)
RPKM normalized coverage (bigwig) files have been generated with 100bp resolution using bamCoverage v3.5.1 (--normalizeUsing RPKM --binSize 100 --smoothLength 300).
Assembly: hg38
Supplementary files format and content: bigwig files (DRIP coverage)
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| Submission date |
Sep 06, 2023 |
| Last update date |
Dec 31, 2023 |
| Contact name |
Lorant Szekvolgyi |
| Organization name |
University of Debrecen
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| Department |
Department of Pharmacy
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| Lab |
Genome Architecture and Recombination Research Group
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| Street address |
Egyetem ter 1.
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| City |
Debrecen |
| ZIP/Postal code |
4032 |
| Country |
Hungary |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE242473 |
DNA methylome, R-loop and clinical exome profile of patients with sporadic Amyotrophic Lateral Sclerosis (DRIP) |
| GSE242475 |
DNA methylome, R-loop and clinical exome profiling of patients with sporadic Amyotrophic Lateral Sclerosis |
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| Relations |
| BioSample |
SAMN37305703 |
| SRA |
SRX21651403 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7764384_hs_drip_K039.bw |
35.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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