CUT&RUN of human: HSPC, GATA2
Briefly, 10,000 cells were captured with BioMagPlus Concanavalin A (Bang Cat. # BP531) for 10 min at room temperature. Permeabilization of cells and binding of primary antibodies were performed oner-night at 4. After washing away unbound antibody with wash buffer, protein A-MNase was added at a 1:100 ratio and incubated for 2 hours. Cells were washed again and placed in a 0-metal block. Then CaCl2 was added to a final concentration of 2 mM to activate protein A-MNase. The reaction was carried out for 30 min and stopped by addition of equal volume of 2XSTOP buffer. The protein-DNA complex was released by centrifugation. DNA was extracted by NEB PCR Cleanup Kit (NEB Cat. # T1030), followed by Qubit fluorometer and bioanalyzer quality control. Protein A-MNase was expressed and purified from BL21(DE) carrying pET-pA-MN. pET-pA-MN was obtained from addgene (Cat#:86973). The CUT&RUN library preparation was performed with DNA library preparation kit (Vazyme, Cat. # ND607) following with the manufactures protocol with minor modifications(4). Briefly, the temperature dA-tailing was decreased to 50C to avoid DNA melting, and the reaction time was increased to 1 hour. After adaptor ligation, 2X volume of DNA Clean beads (Vazyme, Cat. # N411) was added to the reaction to ensure high recovery efficiency of short fragments. After 15 cycles of PCR amplification, the reaction was cleaned up with 1.2X volume of DNA Clean beads. The final libraries were quantified using a VAHTS Library Quantification Kit (Vazyme, Cat. # NQ101) and a D1000 High Sensitivity DNA chip run on a Bioanalyzer 4250 system (Agilent). Two biological replicates per condition were sequenced in a Novaseq 6000 (S4, PE150, Illumina)