Each experimental replicate was a pool of 5 HH23 neural tubes. The neural tubes were dissected and dissociated in Accumax (Innovative Cell Technologies, #AM105) for 30 min at room temperature under mild agitation. After dissociation, the assay was carried out as previously described (Skene and Henikoff, 2017 (link)). Briefly, cells were incubated with BioMag Plus Concanavalin A magnetic beads (Bangs Laboratories, BP531) and rabbit polyclonal anti-H3K27ac antibody (Abcam, cat. Ab177178) overnight at 4°C. Negative control was non-specific IgG and was processed in parallel with experimental samples (anti-IgG (Millipore, cat. #CS200621). After washing, protein A-Mnase was added to a final concentration of 700 ng/mL and incubated for 1 h at 4°C under agitation. The cells were washed and cooled to 0°C. 2 mM CaCl2 was added to activate Mnase for 45 min and terminated by the addition of 2XSTOP buffer (5M NaCl, 0.5M EDTA, 0.5M EGTA, 5% Digitonin, 2.5 µL RNAse A, 20 mg/mL Glycogen). The proteins were digested with proteinase K (10 mg/mL) for 10 min at 70°C and the DNA purified with phenol-chloroform and ethanol precipitation. The DNA was resuspended in water and quantified on Qubit (Invitrogen) with the HS Assay dsDNA kit (Invitrogen, cat. Q32851). Protein A–Mnase was kindly provided by Dr. Steven Henikoff (Howard Hughes Medical Institute, Seattle, EUA).
Biomag plus concanavalin a magnetic beads
Manufactured by Bangs Laboratories
BioMag Plus Concanavalin A magnetic beads are a type of magnetic bead product offered by Bangs Laboratories. They are made from a magnetic core and coated with the lectin Concanavalin A. The primary function of these beads is to bind and capture glycoproteins and glycosylated molecules from complex samples.
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4 protocols using biomag plus concanavalin a magnetic beads
1
H3K27ac ChIP-seq in neural tubes
2
CUT&RUN Analysis of Neural Crest Factors
3
Sp5 Transcription Factor Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Neural folds were dissected from HH7-8 embryos (n = 20 per CUT&RUN experiment). Cells were dissociated in Accumax for 20min at RT under mild agitation. CUT&RUN experiments were carried out as previously described [17 (link)]. Briefly, cells were immobilized on BioMag Plus Concanavalin A magnetic beads (Bangs Laboratories, BP531) and incubated with rabbit anti-Sp5 (Abcam, # ab36593) antibody (1:50) overnight at 4°C. After washing away unbound antibody, protein A-MNase was added to a final concentration of 700ng/mL and incubated for 1h at 4°C. Cells were cooled to 0°C and CaCl2 was added to a final concentration of 2mM to activate the MNase enzyme. MNase digestion was performed for 45min and terminated by the addition of 2XSTOP buffer. The protein-DNA complexes were released by centrifugation and digested with proteinase K for 10 min at 70°C. DNA fragments were isolated via phenol-chloroform extraction and ethanol precipitation. Protein A-MNase was kindly provided by Dr. Steven Henikoff [27 (link)].
4
CUT&RUN Profiling of Active Yap1 and H3K27ac
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