Plasma Cell Transcriptome Profiling

Plasma cells (CD138⁺GFP⁺) were sorted from the bone marrow as described in Fig. S3 A, and plasmablasts were isolated by flow-cytometric sorting as CD22^–CD138⁺GFP⁺ cells after 4 d of LPS stimulation of B cells from the spleen and lymph nodes. Total RNA from in vitro stimulated plasmablasts or ex vivo sorted plasma cells was isolated with the RNeasy Plus Mini Kit (Qiagen), and mRNA was purified by two rounds of poly(A) selection with the Dynabeads mRNA purification kit (Invitrogen). The mRNA was fragmented by heating at 94°C for 3 min in fragmentation buffer. The fragmented mRNA was used as template for first-strand cDNA synthesis with random hexamers and the Superscript Vilo First-Strand Synthesis System (Invitrogen). The second-strand cDNA synthesis was performed with 100 mM deoxy ATP (dATP), dCTP, dGTP, and dUTP in the presence of RNase H, Escherichia coli DNA polymerase I, and DNA ligase (Invitrogen). The incorporation of dUTP allowed for specific elimination of the second DNA strand during library preparation, thereby preserving strand specificity (Parkhomchuk et al., 2009 (link)).