GSM6615864: CUT&RUN II Individual Aptamers_Wnt-_Rep2; Homo sapiens; OTHER
At 24 hr post-transfection, cells were monodispersed by scrapping and pipetting. Nuclei from 5E5 cells were isolated for each reaction. CUT&RUN was performed using the CUTANA CUT&RUN kit (EpiCypher 14-1048), following the kit manual. 500 ng of EZH2 antibody (CST 5246) or Rabbit IgG (EpiCypher 13-0042k) was used in each 50 μL of binding reaction. After chromatin digestion, 0.12 ng of E.coli genomic DNA (kit included) was spiked-in to each reaction with the Stop Buffer. After chromatin release, DNA fragments were purified by Phenol:Chloroform extraction and isopropanol precipitation. Concentrations of DNA were measured using the Qubit 1X dsDNA HS Assay Kit (Invitrogen Q33230). Preparation of Sequencing Library was performed using the NEBNext Ultra II DNA Library Prep Kit (NEB E7103L), and NEBNext Multiplex Oligos for Illumina (NEB E6440S). 6 ng DNA from each CUT&RUN reaction was used as the starting material. Prepared DNA library was dissolved in nuclease-free water, and submitted to GENEWIZ (South Plainfield, NJ, USA) for sequencing. Sequencing was performed on an Illumina HiSeq 4000 sequencer in 2x150 bp mode. Other: CUT&RUN-Seq