GSM4160252: ETO WO ExoT; Mus musculus; OTHER
Single cell suspensions of B cells (20 million per agarose plug) were washed in PBS, embedded in agarose and transferred into plug molds (Bio-Rad CHEF Mammalian Genomic DNA plug kit) and processed as previously described (Canela et al., 2016).END-seq was performed as described in Canela et al., 2017. Agarose plugs were made using the Bio-Rad CHEF Mammalian Genomic DNA plug kit. Embedded cells were lysed and digested using Proteinase K (50º C, 1 hour then 37ºC for 7 hours). Plugs were rinsed in TE buffer and treated with RNase A at 37ºC, 1 hour. The next enzymatic reactions were performed with the DNA in agarose plugs to prevent shearing. DNA ends were blunted for 1 hour at 37C with Exonuclease VII followed by Exonuclease T (NEB) for 1 hour at 25ºC to detect all TOP2 lesions, or only with Exonuclease T (NEB) 1 hour at 25C to detect only DSBs, "ExoT END-seq". After blunting, A-tailing was performed, followed by ligation of “END-seq hairpin adaptor 1”, 5′-Phos-GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGUU[Biotin-dT]U[Biotin-dT]UUACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase. Agarose plugs were then melted and dialyzed and DNA was sonicated to a median shear length of 170bp using Covaris S220 sonicator. DNA was ethanol-precipitated and dissolved in 70 ul TE buffer. Biotinylated DNA was isolated using MyOne Streptavidin C1 Beads (ThermoFisher #650-01), followed by end repair and dA-tailing. The second end was ligated to “END-seq hairpin adaptor 2”, 5′-Phos-GATCGGAAGAGCACACGTCUUUUUUUUAGACGTGTGCTCTTCCGATC*T-3′ [*phosphorothioate bond], using NEB Quick Ligase. Hairpins were digested using USER (NEB), and the resulting DNA fragments were PCR amplified using TruSeq barcoded primer p5, AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATC*T and TruSeq barcoded primer p7, CAAGCAGAAGACGGCATACGAGANNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATC*T, (NNNNNNNN represents barcode and * a phosphothiorate bond). PCR fragments were isolated by size selection from agarose gel, selecting 200-500 bp fragments followed by DNA purification using QIAquick Gel Extraction Kit. Libraries were quantified using KAPA Library Quantification Kit and sequenced using Illumina NextSeq 550.
GEO Accession
GSM4160252