title: GSM2396656: HeLa A KCl; Homo sapiens; OTHER
library_construction_protocol: Cells were grown in 70 ml of DMEM GlutaMAX medium (Gibco), 10% fetal bovine serum (Gibco) in 250 ml Spinner flasks (Bellco Glass Inc.). A total of 5x106 cells were collected by centrifugation for 5 min at 200 ´ g and washed once with PBS. Pellets were resuspended in 2 ml of lysis buffer (75 mM NaCl, 50 mM EDTA, 1% SDS, 20 mM HEPES pH 8.0, 200 µg/ml Proteinase K) and incubated at 42 °C for 30 min. One volume of phenol-chloroform was added to the samples, then mixed and centrifuged at 15000 ´ g for 5 min (4 °C). The water phase was then transferred to a new tube for precipitation with 100 mM NaCl and 1 v of isopropanol. Samples were incubated at -20 °C at least for 1 h. After precipitation, the samples were centrifuged (15000 ´ g, 20 min, 4 °C) and washed with 70% EtOH..Pellets were dissolved in 100 µl of TE buffer. DNA concentrations for library preparations, was measured with a Qubit fluorometric instrument (ThermoFisher Scientific). Patient and control fibroblast cells were grown in DMEM GlutaMAX medium, supplemented with 10% FBS, PEST and 50 µg/ml uridine. Between 4 and 11 ´ 106 cells were collected and total DNA was isolated using the Gentra Puregene Cell Kit (Qiagen) according to the manufacturer´s protocol. Free 5´-ends in mtDNA of HeLa and primary 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 HeLa and primary fibroblast cells were mapped by HydEn-seq by hydrolyzing 1 µg DNA with 0.3 M KOH for 2 h at 55 °C. To calculate the number of ribonucleotides per mtDNA molecule, we treated 1 µg DNA with 10 U of HincII and the digests were purified with HighPrep PCR beads (MagBio), before the KCl or KOH treatment. 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 50-base paired-end sequenced on an Illumina NextSeq500 instrument, to identify the location of the free 5´-ends.
attributes.tag: GEO Accession
attributes.value: GSM2396656