GSM7764385: DRIP, ALS, K485; Homo sapiens; OTHER
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.