GSM4718778: CUT&RUN ESC SUZ12 - rep2; Mus musculus; OTHER
CUT&RUN was performed as described online by Meers and Henikoff at https://www.protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-zcpf2vn. Briefly, cell cultures were harvested, washed and counted with the Countess Automated Cell Counter (Invitrogen). Equal numbers of cells per condition were bound to Concanavalin A-coated magnetic beads (Bangs laboratories), and then permeabilized with 0.015% dig-wash buffer (20 mM HEPES pH 7.5, 150 mM, NaCl, 0.5 mM spermidine, one Roche cOmplete EDTA-free tablet per 50 ml, and 0.015% digitonin). Following bead binding, the supernatant was discarded and the bead-cell slurry was resuspended in 150 µl Antibody buffer (0.015% dig-wash buffer with 2 mM EDTA) containing primary antibody. Antibody incubation was carried out for 2 h at 4 ˚C on a nutator. If needed, bead-cell slurry was further washed with dig-wash buffer and incubated with secondary antibody for an additional hour at 4 ˚C. Samples were washed with Dig-wash buffer to remove unbound antibody, then incubated with Protein A-MNase (700 ng/mL, kind gift of the Sarma lab) in dig-wash buffer for 1 h at 4 ˚C on a nutator. Samples were then washed with dig-wash buffer, resuspended in 100 µL dig-wash buffer and cooled to 0 ˚C on a metal tube rack on ice for 5 min. 2 μL 100 mM CaCl2 was added per sample to activate the MNase, and samples were digested for 30 min at 0 ˚C. The digestion reactions were quenched via addition of 100 μl 2X STOP Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.015% digitonin, 100 ug/mL RNAse A (Thermo Fisher), 50 ug/mL glycoblue (Thermo Fisher), and 100 pg/mL Drosophila S2 spike-in DNA (sonicated to ~200bp). Samples were then incubated for 30 min at 37 ˚C to release CUT&RUN fragments. Supernatant containing CUT&RUN elutions was transferred to fresh tubes and DNA was purified using the MinElute PCR Purification Kit (Qiagen). Purified DNA was quantified using the Qubit dsDNA HS Assay (Thermo Fisher), then used to make sequencing libraries. Paired-end sequencing was performed on a NextSeq 500 (Illumina). Library preparation began from eluted DNA fragments and proceeded as described above starting with the end-repair step. For both RNAseq and CUT&RUN, paired-end sequencing was performed on a NextSeq 500 (Illumina) with a read length of 38 for read 1 and 38 for read 2.
GEO
GSM4718778