title: GSM8263704: mat2a_wt_rep_1; Mus musculus; OTHER
library_construction_protocol: Embryonic stem cells were resuspended in 100μL PBS and and 100 μ Lysis buffer (0.2M Tris-HCl, 0.25M NaCl, 0.025M EDTA, 0.5% SDS, pH 8) were added, next the samples were supplemented with 6 U Proteinase K  and incubated for 40min at 56°C. Further, samples were treated with0,02 U RNaseA  for 10min at room temperature, genomic DNA was isolated using Genomic DNA Clean & Concentrator-10 Kit. Libraries were constructed according to the TOP-seq (Tethered Oligonucleotide-Primed sequencing) protocol (Stasevskij et al., 2017). Briefly, 900 ng of sonicated genomic DNA were end-filled, a 3'-dA mononucleotide extension was added and DNA was ligated to adapters. Ligated DNA was then subjected to Cu(I)-catalyzed cycloaddition reaction in which the alkyne-bearing oligodeoxyribonucleotide with biotin was attached to the azide moiety of the modified DNA. Biotinylated DNA was enriched with streptavidin-coupled magnetic beads and subsequently used in a TOP DNA priming reaction from the attached priming oligodeoxyribonucleotide. The products were amplified and subjected to Ion Proton sequencing.