Flag primary antibody

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)}.

100 ng of Notch chimera constructs were transfected into U2OS cells in a sterile opaque tissue culture-treated 96-well plate (Corning 353296) in triplicate. 24 hr post-transfection, cells were washed once with PBS and fixed using 4% PFA (Thermo Fisher 28906) for 20 min, then washed three times with PBS. Cells were blocked in TBS +5% milk for 1 hr. Then, Flag primary antibody (Sigma-Aldrich F1804) was added 1:250 in TBS +5% milk for 2 hr. Cells were washed three times for 5 min each with TBS +5% milk. The cells were then incubated 1:10,000 with an HRP secondary antibody for 1 hr before being washed five times for 5 min each with TBS. Chemiluminescent substrate was added for 1 min before reading out on a luminescence plate reader.