Hiflex buffer

We collected 5 blood serum samples from 5 mice and used the whole serum from individual mice to isolate EVs as described above. We extracted total RNA including miRs from 5 EV pellets individually using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions with minor modification. Briefly, we added 1.5 volume of 100% EtOH to precipitate the total RNA after chloroform cleaning and eluted the RNA using 13 µL water; 1 µL of the RNA sample was used to measure RNA quality and concentration by Nanodrop (Thermo Fisher Scientific, Waltham, MA, USA) and 12 µL of RNA was used to make cDNA by miScript II RT kit, HiFlex buffer (Qiagen). For qPCR, 20 uL of cDNA was diluted by the addition of 200 uL of water. This diluted cDNA was used to evaluate miRs by RT-qPCR using miRScript SYBR kit (Qiagen) according to the manufacturer’s instructions, using the CFX96 Touch System (Bio-Rad) and the thermal profile suggested by the manufacturer. Expression levels of miR-21-5p and -16-5p were quantitatively compared using the ∆C_t method with mean Ct values of SNORD95, SNORD96 and RNU6-2 as reference genes. These data prove that circulating EVs isolated from a single mouse are enough to evaluate small RNAs.

Total RNA was extracted from cells in culture, or from optimal cutting temperature (OCT) embedded frozen FLC tumor and adjacent normal liver tissue samples using the miRNeasy Mini Kit (Qiagen). The miScript II RT Kit with was used to convert total RNA into cDNA as described by the manufacturer, with use of the HiSpec buffer when used for miRNA and HiFlex buffer when used for mRNA (Qiagen). RNA-Seq libraries were generated using the TruSeq small RNA sample preparation kit (Illumina, San Diego, CA) following the manufacturer's protocol. Libraries were sequenced with 1 × 50 bp single-end reads on an Illumina HiSeq 2000 system. Small RNA reads were mapped and quantified with miraligner and quantified read counts were annotated with Gencode v19. Differential miRNA expression analysis was performed using R version 3.2.3 [78 ] with the Bioconductor package DESeq2 [79 (link)] incorporating variable factors for condition of tumor or normal and patient. For the lncRNA, RNA-Seq libraries were prepared using TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero ribosomal RNA depletion (Illumina). The manufacturer's protocols were used to sequence ribosomal RNA-depleted libraries at 2 × 50-bp paired-end reads on an Illumina HiSEq. 2500 in high-output mode, to an average depth of 86 × 10⁶ paired-end reads per sample (range 43 × 10⁶ to 139 × 10⁶).

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.