title: GSM3543762: Lung tissue_input rep1; Mus musculus; OTHER
library_construction_protocol: DNA fragments harboring 8-oxoG were enriched as previously described (Ding et al. 2017), with minor modifications. Briefly, 5 µg of the genomic DNA extracted from adipose or lung tissues of randomly selected mice (n = 2) were sheared with an S2 ultrasonicator (Covaris, Woburn, MA, USA) in 10 mM Tris buffer (pH 8.0) to obtain ~150-bp fragments. After sonication, the fragmented DNA was concentrated to 20 µl in 100 mM NaPi buffer (pH 8.0) using QIAquick PCR purification kit (Qiagen). A 100-µl volume of 100 mM NaPi buffer containing 20 mM amine-PEG2-biotin (Thermo Fisher Scientific) was added and the mixture was heated to 75 °C for 10 min. After thermal equilibration, 5 mM K2IrBr6 was added for 1 h for 8-oxoG biotinylation. The DNA fragments biotinylated at 8-oxoG were eluted with 125 µl Tris buffer using the QIAquick PCR purification kit and extracted using Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific); strands complementary to those with bound biotinylated 8-oxoG were released by incubation in 150 mM NaOH at 20 °C for 30 min, and concentrated to 10 µl ddH2O using ssDNA/RNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA). The 8-oxoG-enriched DNA fragments obtained by affinity purification were subjected to next-generation sequencing. Libraries were constructed using the TruSeq Nano DNA kit (Illumina, San Diego, CA, USA) and sequenced with the TruSeq SBS Kit v3 on a HiSeq 2000 sequencer (Illumina) to obtain 101-bp paired-end reads.
attributes.tag: GEO Accession
attributes.value: GSM3543762