GSM8698334: mitochondrial DRIP-seq KO input; Mus musculus; ChIP-Seq
Nucleic acid isolation from mitochondria enriched pellets Mitochondria were isolated from the liver of two wild type and two EndoG knock-out animals (Liu JC, et al. Cell Rep. 2016; 16 (6): 1561- 1573). Mitochondrion-enriched samples were resuspended with freshly made 1% formaldehyde in phosphate buffered saline (PBS, 136 mM NaCl, 2.6 mM KCl, 12.4 mM Na2HPO4, 1.7 mM KH2PO4, pH=7.4) supplemented with 5 mM EDTA. Mitochondria were fixed for 10 minutes at room temperature, with shaking (Biosan TS-100 shake; Biosan, Riga, Latvia). Formaldehyde fixation was quenched with the addition of glycine to a final concentration of 0.667 M for 5 minutes at room temperature. Samples were then centrifuged at 10000xg for 10 minutes at 4oC using Eppendorf 5424 centrifuge (Eppendorf, Hamburg Germany). Supernatants were discarded, and pellets were washed in PBS. Lysis was performed using a DRIP-lysis buffer (50 mM TRIS-HCl, 10 mM EDTA, 1% SDS and 0.333 mg/ul Proteinase K (Bioline, Alexandria, NSW, Australia) at 37oC, overnight. Lysates were extracted in phase-lock tubes (5 Prime GmbH, Hilden, Germany) by phenol (50%)-chloroform (48%)-isoamylalcohol (2%) and the nucleic acid content was precipitated by 2.5V 100% ethanol and 10% 3.5 M sodium acetate at -20oC for 45 minutes. After centrifugation with 20000xg for 20 minutes at 5oC, pellets were washed with 70 V/V% ethanol and centrifuged again for 5 minutes at 20000xg. Nucleic acid pellets were dissolved in 10 mM TRIS-Cl pH 7.5. DNA concentration was measured by a NanoDrop-1000 photometer (Thermo Scientific, Waltham, Massachusetts, USA). Isolated nucleic acid samples were fragmented by sonication (Bioruptur Plus; Diagenode, Belgium; 10 cycles: 30 s “on” followed by 30 s “off”) to an average fragment size of 300-500 bp. 2% of the sonicated material was kept as 'input'. The remaining samples were divided into two aliquots that were or were not treated with E. coli RNaseH New England Biolabs, Ipswich, Massachusetts, USA). RNaseH digestion (was performed at 37oC, overnight by adding 7U of enzyme for 1ug of measured DNA content. RNaseH-treated samples were then PCR cleanup purified (Macherey-Nagel, Düren, Germany) and eluted in 5 mM TRIS-Cl pH 7.5 solution. RNA-DNA hybrid immunoprecipitation was performed using the S9.6 mouse monoclonal antibody (HB-8730, ATCC). Nucleic acid content of the IP and input samples was measured by a Qubit fluorometer (Thermo Fisher Scientific). DRIP 'IP' and 'input' samples and RNaseH1-treated (negative control) DRIP samples were concentrated to less than 30 l volume with Eppendorf Concentrator Plus (Eppendorf, Hamburg Germany) at 30oC for 30 minutes. Illumina compatible sequencing libraries were generated from fragmented 1 – 10ng of dsDNA using Ovation® Ultralow System V2 (Nugen, San Carlos, CA, USA) library preparation kit according to the manufacturer's protocol. Briefly, fragmented dsDNA samples were end-repaired and A-tailed, then Illumina compatible indexed adaptor were ligated. Adaptor ligated dsDNA were amplified, and the products were purified using MagSi-DNA NGSPREP Plus (MagnaMedics GmbH, Aachen, Germany) magnetic beads. Sequencing libraries were quantified using Qubit fluorometer (Thermo Fisher Scientific), the fragment size distributions were analyzed on Agilent BioAnalyzer 2100 using DNA High Sensitivity assay (Agilent Technologies, Santa Clara, CA, USA). Sequencing libraries were normalized to the same molar concentration and pooled together. Library pool was sequenced on NextSeq500 instrument (Illumina, San Diego, CA, USA) generating single read 75bp long sequencing reads. Fastq files were generated automatically after the sequencing run by Illumina BaseSapce. The library preparations and the sequencing run were performed by Genomic Medicine and Bioinformatics Core Facility of Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, Hungary.