title: CUT&RUN for Zeb2, T-bet and IgG control in live mouse age-associated B cells
design: CUT&RUN for Zeb2, T-bet and IgG control in live mouse age-associated B cells
library_construction_protocol: bm12-cGVHD lupus model was induced following JOVE protocol (10.3791/53319). Briefly, 8-12-week-old mice were intraperitoneally injected with 10 million CD4+ T cells from bm12 mice purified by magnetic cell sorting and mice were sacrificed for analysis after 2 weeks. Single cell suspensions of splenocytes from Bm12-induced lupus T-bet reporter mice were subjected to red blood cell lysis and filtered through 70 um nylon mesh. Cells were stained for surface markers under instructions. age-associated B cells were collected through flow cytometry. 0.5 million B cells were incubated with CoA-coated magnetic beads, and then primary antibodies (anti- Zeb2, anti-T-bet, anti-IgG)were added to incubate for 1 hour at room temperature. After incubation, secondary antibodies and pG-Tn5 adapter complex were added. Then,tagmentation took place for 1 hour, DNAs were extracted and purified, and PCR amplified into a final CUT&RUN library CUT & RUN libraries were generated following instructions of the manufacturer's protocol (CST, DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN),##56795)