Sample 2
Danio rerio
Protocols: Zebrafish embryos of the AB strain were collected and the yolk was removed. Cells were dissociated into single cells with trypsin and collagenase.Dissociated single cells were combined with CoA beads and incubated with primary antibody anti-pStat3 (HUABIO, 1:50) or anti-pSmad1/5 (Sigma-Aldrich, 1:50) overnight. Samples were incubated with secondary antibody Goat anti-rabbit (Bio-Rad, 1:100), conjugated with transposase, and fragmented.  DNA was purified using Tagment DNA extraction beads provided in the NovoNGS® CUT&Tag® 2.0 High-Sensitivity Kit (for Illumina®）from Novoprotein Scientific Inc., Shanghai, China. The nucleic library was constructed using primers and reagents provided with the NovoNGS® CUT&Tag® 2.0 High-Sensitivity Kit (for Illumina®）from Novoprotein Scientific Inc., Shanghai, China.  Take out the cryovial and shake it in 37°C water bath to quickly thaw it within 1-2min.Cells were centrifuged for 5 min at 600×g at room temperature. Cell activity was detected with LUNA-FLTM and counted. CUT&Tag assay was performed as described previously(Kaya-Okur, Wu et al. 2019). Briefly, the cells are bound to Concanavalin A-coated magnetic beads, and the cell membrane is permeabilised by Digitonin. The enzyme pA-Tn5 Transposase precisely binds the DNA sequence near the target protein under the antibody guidance and results in factor-targeted tagmentation. DNA sequence is tagmented, with adapters added at the same time at both ends, which can be enriched by PCR to form the sequencing-ready libraries. After the PCR reaction, libraries were purified with the AMPure beads and library quality was assessed on the Agilent Bioanalyzer 2100 system.
strain
AB
organism
Danio rerio
organism
Danio rerio
collection date
not collected
sex
not available
scientific_name
Danio rerio
common name
zebrafish
organism part
animal pole
developmental stage
cleavage stage
genotype
wild type genotype
isolate
not applicable
age
3
geographic location (country and/or sea)
not collected