DN-3
Mus musculus
Protocols: bm12-cGVHD lupus model was induced following JOVE protocol. 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. DP and DN B cells were collected through flow cytometry. Briefly, 50,000 live cells were freshly isolated and resuspended in lysis buffer (10 mM Tris·Cl, (pH 7.4) ,10 mM NaCl，3 mM MgCl2，0.1% (v/v) Igepal CA-630) and pelleted. The cell pellet was resuspended in transposase reaction mix (5×TTBL 10μl, TTE Mix V50 5μl, ddH2O 35μl) and incubated at 37°C for 30 min. Fragmented DNA was purified using VAHTS DNA Clean Beads and library was generated by PCR.