CUT&Tag of Sus scrofa:14d male testis
CUT&Tag Library Preparation: (1) ConA beads (concanavalin A-coated magnetic beads) were treated with Binding Buffer; cells were harvested and counted at RT, then incubated with ConA beads.(2) Primary antibodies were added (designed to penetrate membranes/nuclear pores for intranuclear target binding).(3) Secondary antibodies were introduced to enhance pG-Tn5/pA-Tn5 transposon targeting.(4) Hyperactive pG-Tn5/pA-Tn5 Transposon was added.(5) Tagmentation buffer activated transposase-mediated DNA fragmentation at target sites.(6) DNA was extracted, PCR-amplified, and purified using magnetic beads.(7) Library concentration was quantified via Qubit/PicoGreen fluorescence.(8) Fragment size distribution was assessed using Agilent 2100/4200 Bioanalyzer or 2% agarose gel electrophoresis.(9) Sequencing was performed on NovaSeq/DNBSEQ-T7 platforms (PE150).