Ssoadvanced preamp supermix

gDNA extracted from tumor tissue and cells, and cfDNA extracted from CSF and plasma, was pre-amplified at CN using Q5 hot start high-fidelity master mix (New England Biolabs), and at NU using SsoAdvanced PreAmp Supermix (Biorad), with 50 nmol/L each of forward and reverse primer. Pre-amplification at CN was performed using Assay A primers (Supplementary Fig. 1) in ABI 2720 thermocycler: 98 °C for 3 min; nine cycles of 98 °C for 10 s, 58 °C for 3 min, 72 °C for 30 s; and an extension of 72 °C for 2 min. Product was diluted 1:5 with TE buffer (pH 8.0). Pre-amplification at NU was performed on the BioRad T100 thermocycler using the following conditions: 95 °C for 3 min, 10 cycles of 95 °C for 15 s, annealing temperature (58 °C) for 4 min. The pre-amplified product was diluted 1:5 with molecular grade water. At CN, 0.025 ng gDNA from DMG-51-T was used as a positive control per a previously established institutional protocol^{17 (link),19 (link)}. 2 ng of tumor gDNA was used for ddPCR analysis of patient-matched tumor, CSF and plasma/serum specimens. Where applicable, starting cfDNA aliquots were speed-vacuum concentrated from 100 µL to 10.5–11 µL prior to pre-amplification. Assay A primers were used for ctDNA pre-amplification of all samples at CN, while Assay C primers were used for PCR pre-amplification at NU^{18 (link)}.

cDNA was generated from RNA samples from FACS sorted cells with the iScript Reverse Transcription Supermix (#1708840 Bio-Rad, Hercules, CA, USA), N = 6 per condition. Target-specific preamplification was performed on cDNA generated from RNA samples using SsoAdvanced PreAmp Supermix (#1725160 Bio-Rad) containing Sso7d fusion polymerase. Briefly, 20 μl of cDNA was pre-amplified in a total volume of 50 μl containing 25 μl of 2× SsoAdvanced PreAmp Supermix and 21 primer pairs, 50 nM of each primer. Preamplification was performed at 95°C for 3 min followed by 12 cycles of amplification at 95°C for 15 s and 58°C for 4 min. The samples were moved directly to ice and stored at −80°C. Pre-amplified cDNA was diluted 1:5 with H₂O. Controls performed for pre-amplification include qPCR and gel electrophoresis analysis of RNA extracted from both whole DRG tissue and FACS isolated DRG neurons from naïve mice. Following qPCR and gel analysis, all pre-amplified samples had no double bands and ran at expected weights based on product size.

Total exosomal RNA was extracted by a commercial kit (PureLink RNA Micro kit; Invitrogen, Carlsbad, California, USA). According to the manufacturer’s instructions, RNA was extracted, purified, and eluted in a final volume of 15 µl and stored at −80°C.
Synthesis of cDNAs was carried out using a commercial kit (iScript Advanced cDNA synthesis kit for RT-PCR; Biorad, Hercules, California, USA) and a preamplification step was included (obtained by the use of SsoAdvanced PreAmp Supermix; Biorad, Hercules, California, USA) to improve mRNA detection and measurement. Preamplification primer mix included the genes HSD11B2 and β-2 microglobulin (B2M).

To extract RNA from Trizol stocks we used Direct-zol™ RNA MicroPrep (ZymoResearch, USA, Cat. No. R2062) according the manufacturer’s protocol. The total RNA was extracted in 10 ml, measured using Nanodrop-2000, and tested with Experion™ RNA HighSens Analysis Kits (Bio-Rad, Cat. No. 700-7105) using the Experion™ Automated Electrophoresis System (Bio-Rad). 5 ml of total RNA from either melanocytes or iridophore (50-60 ng/ml) was used directly for NanoString expression analysis.
cDNA was synthesized using 1 μl of total RNA from melanocytes or iridophores with iScript™ Advanced cDNA Synthesis Kit (Bio-Rad, Cat. No. 1725038) following by 6 cycles of pre-amplification with pooled 47 pairs of MTE primers designed for NanoString CodeSet (SsoAdvanced™ PreAmp Supermix, Bio-Rad, Cat. No. 1725160); amplified dsDNA was analysed using the NanoString expression protocol.

Before transcription into cDNA using the iScript gDNA Clear cDNA Synthesis Kit (Biorad, Hercules, CA, USA), RNA was digested with DNase to remove genomic DNA contamination. Then, cDNA was pre-amplified with SSoAdvanced PreAmp Supermix (Biorad) using a pool of primers as listed in Table 2. qPCR was carried out with ABI Prism 7500 Fast RT-PCR System (Applied Biosystems, Waltham, MA, USA) cycler as previously described [31 (link)] and run over 40 PCR cycles. Gene expression was confirmed positive when both duplicates of each sample revealed detectable Ct (cycle threshold) values (≤39). For method establishment and validation of gene expression, we conducted the same protocol using 2.5 ng RNA from immortalized cell lines for pre-amplified cDNA. Primer sequences for qPCR were listed in Table 2. Ct levels from genes of interest (GOI) were normalized with TBP using the mathematical model ratio 2^−ΔCt (dCt = Ct gene of interest−Ct TBP).

Expression of genes found modulated in the microarray study was analyzed by quantitative PCR (qPCR). Complementary DNA (cDNA) was synthetized by reverse transcription of total RNA in presence of oligo(dT) and Transcriptor Reverse Transcriptase (Invitrogen, Thermo Fisher Scientific, USA). cDNA was then amplified using SsoAdvanced PreAmp Supermix (BIORAD). Q-PCR was performed using the LightCycler system (Roche Molecular System Inc., USA) and SYBR Green reagent mix (Ozyme, France) according to suppliers’ instructions. Primer sequences are detailed in Table S4. Glyceraldehyde-3-phosphate- dehydrogenase (GAPDH), beta-2- macroglobulin (B2M) and ribosomal protein S28 (RPS28) mRNA were quantified in each sample and used for normalization using Genorm application. For each volunteer, gene modulation was defined as the ratio between the level of mRNA expression in AL versus the level of mRNA expression in NL sample. For each gene, expression levels in AL samples versus expression levels in non-lesional samples were compared using Wilcoxon matched-pairs signed rank test (p < 0.05).