Rneasy minielute

Freshly sorted primary cells were lysed in RLT buffer (Qiagen, Crawley, West Sussex, UK) and stored at −80°C. Total RNA was extracted using an RNeasy MiniElute or MicroElute Kit (Qiagen), according to the manufacturers’ instructions. qrtPCR reactions were performed as previously described use Taqman probes (see Additional file 2) [14 (link)]. Results either were calculated using the ΔΔCt method and expressed as the mean fold gene expression difference in three independently isolated cell preparations over a comparator sample with 95% confidence intervals, or, for single cell experiments, presented as raw 1/Ct values.

Total RNA was purified using AllPrep DNA/RNA Mini and RNeasy MiniElute kits (Qiagen) according to the manufacturer’s instructions. Purified RNA quality was evaluated using Agilent RNA 6000 Pico Kit. Samples were then either supplemented with RNase inhibitor (RNase OUT, Thermofisher Scientific) and stored at -80°C or taken directly to reverse transcription.

Purified in vitro ligated product was used as template for the in vitro transcription by mMESSAGE mMACHINE T7 Transcription Kit (Ambion) according to the manufacturer’s protocol. For N mRNA production, we amplified the N protein-coding region by PCR (sense: GGC ACA CCC CTT TGG CTC T; antisense: TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TTT TCT AGG CCT GAG TTG AGT CAG CAC) with phCMV-N as template. Then the purified PCR product was used as a template for in vitro transcription by mMESSAGE mMACHINE T7 Transcription Kit as described above. RNA was purified by RNeasy mini Elute (Qiagen), eluted in nuclease-free water, and quantified by UV absorbance (260 nm).