RNA sequencing of single LN cells sorted from virus-activated CD4 T clusters

PA-GFP mice were infected subcutaneously with either rVSV or rLCMV viruses. Two days upon infection, draining popliteal lymph nodes (LN) were photoactivated in areas containing Ag-specific CD4+ T cell clusters. Photoactivated cells were single-cell sorted and RNA sequencing performed to determine cell composition of activated CD4+ T cell clusters. CFP+ Ag-specific CD4 T cells were transfered into PA-GFP mice, which were then infected in the footpad with either rVSV or rLCMV viruses. Two days upon infection, draining popliteal lymph nodes (LN) were photoactivated in areas containing Ag-specific CD4+ T cell clusters. LN were processed and photoactivated GFP+ cells were single-cell sorted in 384-well capture plates containing 2 μL of lysis solution and 10 barcoded poly(T) reverse-transcription (RT) primers for single-cell RNA-seq