title: GSM7111608: CnR4_H3K4me1; Homo sapiens; OTHER
library_construction_protocol: For Cut&Run sequencing, cells were washed twice and then coupled to ConA-coated magentic beads (Polysciences Europe). Cells were resuspended with antibody buffer (0.005% digitonin and 2 mM EDTA). Then cells were incubated with the antibody (see for details above) for 2 hours at RT. Cells were then washed in the presence of digitonin and 700 ng/ml MNase was added per sample for 1 hour at 4°C, followed by two more washes. Following the initial washes, the MNase treated cells were washed once in low salt rinse buffer and then incubated in buffer containing CaCl2 and digitonin to activate the protein A-MNase. The DNA was digested for 30 min on ice and the Mnase activity was then stoped in the presence of 50 µg/ml Rnase A. DNA fragments were released at 37°C for 30 min and the decrosslinking was done for 1 hour at 50°C in the presence of Proteinase K. DNA was extracted wit phenol-chloroform and DNA was precipitated with ethanol and resuspended in TE buffer. In some cases (Cut&Run samples where antibodies were used for H3K4me1, H3K4me3, H3K27ac and H4ac), a 1 min crosslinking with 1% formaldehyde was performed. In these cases, all buffers were used without digitonin and instead 0.05% SDS and 0.1% Triton X-100 were added to the wash buffer. The DNA libraries were prepared with 6 ng of CUT&RUN DNA fragments, using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. The libraries were PCR-amplified with 15 PCR cycles using the NEBNext Multiplex Oligos for Illumina Kit. The libraries were purified using Agencourt AMPure XP beads (Beckman Coulter). Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in single-read or paired-end mode on a NextSeq500 or NextSeq2000 platform (Illumina). followed by sequencing.