GSM2776465: Fibroblast KCl; Homo sapiens; OTHER
Approximately 5 × 106 cells were collected and lysed for 30 min at 42 °C in lysis buffer (10 mM Tris-HCl pH 8.0, 5 mM EDTA, 10 % SDS). An equal volume of phenol-chloroform was added, samples were mixed and centrifuged (15,000 g for 5 min, 4 °C). The aqueous phase was saved and 100 mM NaCl and one volume isopropanol were added. After precipitation at -20 °C for one hour, the samples were centrifuged (15,000 g, 20 min, 4 °C) and the pellets were washed with 70 % EtOH. The DNA was resuspended in 100 μl TE buffer. DNA concentrations were measured using the Qubit fluorometric instrument (ThermoFisher Scientific).  Free 5´-ends in mtDNA of fibroblast and patient-derived fibroblast cells were mapped by 5´-End-seq by treating 1 µg DNA with 0.3 M KCl for 2 h at 55 °C. RNA residues in mtDNA of fibroblast and patient-derived fibroblast cells were mapped by HydEn-seq by hydrolyzing 1 µg DNA with 0.3 M KOH for 2 h at 55 °C. After ethanol precipitation, the DNA fragments were treated for 3 min at 85 °C, phosphorylated with 10 U of 3′-phosphatase-minus T4 polynucleotide kinase (New England BioLabs) for 30 min at 37 °C, heat inactivated for 20 min at 65 °C and purified with HighPrep PCR beads (MagBio). Phosphorylated products were treated for 3 min at 85 °C, ligated to oligo ARC140 overnight at room temperature with 10 U of T4 RNA ligase, 25% PEG 8000 and 1 mM CoCl3(NH3)6, and purified with HighPrep PCR beads (MagBio). Ligated products were treated for 3 min at 85 °C. The ARC76–ARC77 adaptor was annealed to the second strand for 5 min at room temperature. The second strand was synthesized with 4 U of T7 DNA polymerase (New England BioLabs) and purified with HighPrep PCR beads (MagBio). Libraries were purified, quantified with a Qubit fluorometric instrument (ThermoFisher Scientific) and 75-base paired-end sequenced on an Illumina NextSeq500 instrument, to identify the location of the free 5´-ends.