Transcriptomics of THP-1 macrophages after treatment with NLRP3-induced EVs

Inflammasome activation in macrophages induces the release of EVs, however, the effect of these inflammasome-induced EVs on recipient cells is poorly characterized. To characterize the effect EVs released upon LPS + nigericin stimulation, we performed 3' sequencing on the recipient cells (NLRP3 KO THP-1 macrophages and NLRP3 KO THP-1 macrophages that have been reconstituted with NLRP3 to resemble the WT). As controls, RNA isolated from EVs themselves or LPS- or nigericin-treated cells were subjected to 3' sequencing. Five replicaes of each sample were generated, i.e. 100 samples were transferred to the Next Generation Sequencing Core Facility of the Medical Faculty of the University of Bonn for library preparation using the QuantSeq 3’ mRNA Library Prep Kit FWD for Illumina according to the manufacturer’s instructions. Samples were processed in three batches. RNA libraries were prepared with 50 ng of total cellu-lar RNA or 30 ng of total vesicular RNA. We performed 3’ sequencing on the Illumina Hiseq2500 with an average of 20 · 106 reads per sample.