GSM9393707: Input control 15ss rep2; Danio rerio; ChIP-Seq
At 20-22 ss, embryos (50-100 per library) were homogenized and fixed by formaldehyde crosslinking, quenched, and nuclei were isolated. Chromatin was sheared using a Bioruptor Pico (Diagenode; 4 cycles of 30 s ON/30 s OFF) to around 200-500 bp fragments. After preclearing with Dynabeads Protein A (Life Technologies), biotinylated Sox10‑DNA complexes were captured using Dynabeads Streptavidin M‑280 (Invitrogen). One‑twentieth of the sample was reserved as input DNA. Beads were washed under stringent conditions, crosslinks were reversed, and DNA was purified. ChIP and input DNAs were prepared into sequencing libraries using the MicroPlex Library Preparation Kit v3 (Diagenode) following the manufacturer's instructions. PCR amplification was 14 cycles for ChIP and 12 cycles for input libraries. Libraries were sequenced on an Illumina NovaSeq with 150‑bp paired‑end reads. Cut&Run was performed as previously described by Skene and Henikoff, 2017, with minor modifications. A total of 150 embryos were collected at 16 somite stage. The chorion was removed for all embryos prior to dissociation in 500 µL Accumax for 25 min. Cells were pelleted at 600x g for 3 min at room temperature, washed twice in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1× protease inhibitor tablet), and resuspended in 800 µL of wash buffer. Cells were bound to 80 µL of Concavalin-A beads for 10 min with rotation and distributed into 100 µL aliquots for the antibody condition. Two replicates per condition were included for ELF1 and IgG controls. The samples were collected on a magnetic stand and incubated with 100 µL of antibody buffer (wash buffer with 0.05% digitonin and 2 mM EDTA) containing primary antibodies (1:50 dilution; ELF1 bs-7042R, Bioss) for 12 h at 4°C on a thermomixer with 350 rpm. The samples were spun quickly 100×g, beads were collected using the magnetic stand, washed twice with 200 µL cell permeabilization buffer (wash buffer with 0.05% digitonin), resuspended in 50 µL of cell permeabilization buffer containing 2.5 µL of pAG-MNase fusion protein (700 ng/mL) and incubated 1 hour at 4°C on thermomixer with 350rpm. Samples were washed twice more, resuspended in 100 µL of same buffer, chilled on ice to 0°C, and digestion was initiated by adding 2 µL 100 mM CaCl2 for 1 h at 1°C on a thermomixer with 350 rpm. The reaction was stopped with 50 µL Stop Buffer (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 0.05% Digitonin, 100 μg/ml RNase A, 50 μg/ml Glycogen, 1:100 E. coli DNA spike-in) and incubated at 37°C for 10 min to release CUT&RUN fragments from insoluble chromatin. To collect the fragmented antibody-bound DNA, beads were collected by placing samples on the magnet and the supernatant was transferred to new low-binding tubes. Chromatin proteins were digested with 1 µL 20% SDS and 2.5 µL proteinase K (20 mg/uL) for 10 min at 70°C. DNA was purified by 300 µL phenol:chloroform extraction, followed by 300 µL chloroform cleanup. The aqueous (upper) layer was transferred to fresh tubes containing GlycoBlue (2 mg/mL). To precipitate DNA, 750 µL of 100% ethanol was added and incubated for 12 h at -80°C. Samples were centrifuged by 20 min 16,000×g and resuspended in 15 µL Nuclease-Free Water. DNA concentrations were measured with the Qubit fluorometer, and sequencing libraries were prepared.