title: GSM8698086: Endoderm_Donor_rep2_IgG; Xenopus laevis; OTHER
library_construction_protocol: CUT&RUN was performed according to Phelps W et al. eLife (2023) with some modifications. Endoderm was collected from 25 neurula-stage embryos (NF stage 18) per sample. For nuclear isolation, the endoderm tissues were incubated in Newport 2.0 buffe (Briggs et al. 2016) to facilitate tissue dissociation, by gently agitating the supernatant but not disturbing the tissue. The nuclei were extracted by resuspending in 1 ml ice-cold nuclear extraction buffer (20mM HEPES-KOH, pH 7.9, 10mM KCl, 500 μM spermidine, 0.1% Triton X-100, 20% glycerol), and subsequently resuspended in 600 µl nuclear extraction buffer. 150 µl concanavalin A beads (Epicypher, 21-1401) were resuspended in 850 µl binding buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1mM MnCl2) per sample and activated by washing twice with 1 ml binding buffer. The nuclei were added to 300 µl bead suspension under gentle vortexing and incubated for 10 min at RT while rotating to allow binding of the nuclei to the beads. The supernatant was discarded and the nuclei were incubated in 1 ml blocking buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA, 2 mM EDTA,1x protease inhibitor)  for 5 min at RT, then incubated in 1:100 primary antibody solution overnight at 4°C and washed twice using 1 ml wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.1% BSA, 1x protease inhibitor). To facilitate pAG-MNase (Epicypher, 15-1116) binding to the primary antibody, samples were incubated under rotation at 4°C for 1 h, washed twice using wash buffer, and resuspended in 150 µl wash buffer. To activate the MNase digestion reaction, 3 µl 100 mM CaCl2 was added to each sample and incubated at 0°C for 30 min, then quenched using 2x STOP buffer (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/mL RNase A, 40 μg/mL glycogen), containing 1 ng exogenous spike-in DNA (E. coli, Epicypher 18-1401). To release chromatin fragments, the samples were incubated at 37°C for 20 min, following centrifugation at 16.000 g for 5 min at 4°C. The supernatant containing soluble chromatin fragments was mixed with SDS and proteinase K (Sigma-Aldrich) at 70°C for 10 min and subjected to on-column DNA purification (QIAGEN, MiniElute PCR Purification Kit). CUT&RUN sequencing libraries were prepared using NEB Ultra II DNA library prep kit (NEB, #E7645), following the manufacturer's instructions, using 12 PCR amplification cycles with CUT&RUN-specific cycling parameters (45 s 98°C for polymerase activation, 14 cycles of 15 s 98°C DNA melting and 10 s 60°C primer annealing and short extension, 1 min 72°C final extension), without size selection. The libraries were sequenced on a NovaSeq X+ sequencing platform.