CUT&RUN Profiling of Histone Deacetylases in Forelimb Development

Experiments were performed according to EpiCypher’s CUTANA CUT&RUN protocol (EpiCypher #15-1016) with the following modifications. 200,000–300,000 cells from E9.25 (21–23 S) forelimbs and anterior E10.5 (32–35 S) forelimbs were incubated overnight at 4 °C on a nutator with 1:250 FLAG primary antibody (Sigma #F3165), 1:100 HDAC1 (Abcam ab7028) or 1:200 HDAC2 (Abcam ab7029). The following day, samples were incubated at room temperature for 30 min. with secondary antibody, 1:100 Donkey anti-mouse (Jackson ImmunoResearch #715-035-150) or Donkey anti-rabbit (Jackson ImmunoResearch #711-005-152), followed by three washes in Digitonin wash buffer. CUTANA pAG-MNase was then incubated with samples for 10 min at room temperature and then the MNase reaction was performed for 2 h at 4 °C on a nutator. Libraries were generated using NEBNext Ultra II DNA Library Prep Kit with 14 PCR cycles and cleaned up to remove adapters using AMPure XP beads (Beckman Coulter) or SparQ PureMag beads (QuantaBio). Samples were sequenced on an Illumina NextSeq 500 instrument using PEx75 to a depth of depth of 3–5 million reads. Peaks were called using MACS^{79 (link)}.