title: GSM8672971: pituitary gland, Bmpr2a_rep2_IgG; Danio rerio; OTHER
library_construction_protocol: Nuclei were harvested from a mass of zebrafish pituitary cells. Nuclei were washed and incubated with 10 μl concanavalin A-coated magnetic beads. Bead-bound cells were then resuspended with dig-wash buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM spermidine; 1X protease inhibitor cocktail; 0.05% digitonin; and 2 mM EDTA) and a 1:50 dilution of primary anti-phospho-Smad1/5/9 antibody (Cell Signaling Technology, 13820, Rabbit, USA) or control IgG on a rotating platform overnight at 4 °C. The beads were washed in dig-wash buffer, and the primary antibody was removed using a magnetic stand and then incubated with a secondary antibody (1:100) for 60 min. After incubation, the beads were washed in dig-Hisalt buffer and incubated with the proteinA-Tn5 adapter complex for 1 h. Next, the cells were resuspended in Tagmentation buffer (10 mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. The DNA fragments were purified using phenol‒chloroform-isoamyl alcohol extraction and ethanol precipitation. The sequencing libraries were prepared according to the manufacturer's instructions and cleaned using XP beads (Beckman Counter)