title: MinION sequencing
library_construction_protocol: RNA from FACS acquired cells was isolated using ArcturusTM PicoPureTM RNA Isolation Kit (AppliedBiosystemsTM, KIT0204) following guidelines for RNA isolation from Cell Pellets with on-column DNAse treatment (Qiagen, 79256), with some modifications. 20000 cells were sorted out of muscles, then pooled from 3 mice directly to 250 μl extraction buffer. During precipitation step we used 1:1 (v:v) 70% ethanol:extraction buffer ratio. RNA was eluted in 15 μl elution buffer in every case.  Additional amplification step of mRNA-1273 (applicable for samples with expected low prevalence of vaccine mRNA, like cells sorted from vaccine injection sites) was applied as modification to standard SQK-PCB111-24 (Oxford Nanopore Technologies) protocol. After synthesis of the first cDNA strand, samples from the reverse transcription reaction were preamplified in a 12-cycle PCR reaction with RTP and SSPII_Mod_2 (specific for mRNA-1273, with UMI sequence: TTTCTGTTGGTGCTGATATTGCTTTVVVVTTVVVVTTVVVVTTVVVVTTTCCACCGACAACACCTTCGTGAGCGG) primersusing LongAmp Hot Start Taq Master Mix. The reaction products were then purified on Kappa beads, washed with SFB buffer, eluted with H2O and used for the subsequent PCR reaction with barcoded primers, performed according to the SQK-PCB111-24 protocol (14 cycles, 5 min amplification). Finally, cDNA concentrations were measured on Qbit and 12 libraries containing a total of ~60 ng cDNA were used for ligation with the RAPT adapter and sequenced. Sequencing was performed with MinION device (Oxford Nanopore Technologies, Flow cell type FLO-MIN106; RevD).