Single-cell Preparation and Transcriptional Analysis

Spleen, MLN, and PP cells were made into single-cell suspensions in complete IMDM and prepared for FACS analysis or sorting. Red blood cells were lysed using ACK lysis buffer (Thermo Fisher Scientific). For qRT-PCR, purified cells and tissue were harvested in RLT lysis buffer (QIAGEN) or RNAlater (Thermo Fisher Scientific). Tissues were homogenized, RNA was extracted, and cDNA was generated as previously described (Pelly et al., 2016 (link)) or with miSCRIPT II and HiFlex buffer (QIAGEN) for miRNA expression analysis. cDNA was amplified and normalized to the house-keeping gene Hprt or rnu6b (Invitrogen) for miRNAs and expressed as fold-change (as indicated in the figure legends). The sequences for primers used are listed in Table 1 or are previously published (Pelly et al., 2016 (link)). miRNA primers were obtained from QIAGEN.