GSM5775778: A673 ETV6 dTAG cells - 24 hour DMSO treated - ETV6 HA-tag CUT&Tag with Abcam anti-body replicate 2; Homo sapiens; OTHER
CUT&Tag (Cleavage Under Targets and Tagmentation) was performed as described (Kaya-Okur, et al., Nature Cummunications, 2019) with slight modifications by the Lessnick Laboratory (Nationwide Children's Hospital, Columbus, OH). 250,000 cells per CUT&Tag condition were bound to BioMag® Plus Concanavalin A-coated magnetic beads (Bangs Laboratories, BP531) and incubated with primary antibodies (ETV6 rabbit, Bethyl A303-674, 1:50; ETV6 mouse, Sigma WH0002120M1, 1:50; Rabbit anti-mouse, Abcam, ab46540, 1:50) overnight at 4 °C and secondary antibodies (guinea pig anti-rabbit IgG, Antibodies-Online ABIN101961, 1:100; Rabbit anti-mouse, Abcam ab46540, 1:100) for 1 hour at room temperature. Adapter-loaded Protein A-Tn5 fusion protein was added at a dilution of 1:250 and incubated for 1 hour at room temperature. To activate the Tn5, tagmentation buffer containing MgCl2 was added and samples were incubated for 1 hour at 37°C. Reactions were stopped by addition of EDTA and DNA was solubilized with SDS and Proteinase K for 1 hour at 50°C. Total DNA was purified using Phenol/Chloroform extraction followed by Ethanol precipitation. CUT&Tag libraries were prepared with NEBNext HiFi 2x PCR Master Mix (NEB M0541S) and indexed primers10 using a combined Annealing/Extension step at 63°C for 10 seconds and 15 cycles followed by a 1.1X post-amplification AMPure XP (Beckman Coulter, A63880) bead cleanup. The fragment size distributions and concentrations of the final libraries were determined using the High Sensitivity D1000 Screen tape assay (Agilent, 5067-5584 and 5067-5585) on the Agilent 2200 TapeStation System. Libraries were pooled and sequenced (2 x 150 bp paired end) on the Illumina HiSeq4000 platform (Nationwide Children's Hospital Institute for Genomic Medicine). Two independent replicates of each CUT&Tag sample were prepared.