Qx200 droplet reader

FACS-purified adult cochlear SCs were collected from P60 Lfng^EGFP mice as detailed previously. RNA was isolated from 30,000 GFP-positive and GFP-negative cells with each assay standardized to 2,000 cells per sample collected in triplicates to use for the quantification experiment. The ddPCR Droplets were generated using the QX200 AutoDG Droplet Digital. PCR was performed as described in the QX200^TM ddPCR^TM EvaGreen^® Supermix instructions². Droplets were read with a QX200^TM Droplet Reader (BioRad) and analyzed with QuantaSoft software (BioRad). Primer sequences utilized for ddPCR are provided in the Supplementary Table S3.

For each patient, 500 μl of blood was collected in an EDTA microtainer tube. After 5 min of centrifugation, 200 μl of plasma was separated from the blood cell pellet and replaced by an equivalent amount of 1X PBS, pH 7.4. Using the whole blood DNA extraction kit from Qiagen, DNA was isolated from the PBS resuspended blood cell pellet and total DNA concentration was measured by NanoDrop (Thermo Fisher Scientific). We used digital droplet PCR (ddPCR) to determine EBV load in each sample by amplifying EBV BALF5 and human β-actin gene, using primers and probes shown in Table 1. The duplex ddPCR reactions were prepared in a total volume of 20 μl which contained 10 μl of ddPCR Supermix for probes (No UTP) (Bio-Rad Laboratories), and 2 sets of each primer and probe combination (0.9 μM of primers and 0.25 μM of probes). The BioRad Automated Droplet Generator (AutoDG) (Bio-Rad Laboratories) was used to ensure consistent droplet generation. After the ddPCR reaction, the number of positive and negative droplets were counted by the Bio-Rad QX200TM Droplet reader and EBV viral loads quantified as copies/ng human DNA.

DNA was extracted from frozen blood samples with MasterPure DNA Purification Kit for Blood Version II (Epicenter). The DNA quality was tested using a Qubit 2.0 Fluorometer (Invitrogen). Primers and assays for droplet digital PCR (ddPCR) of the fragments were designed by the tool on the BioRad website and by the Primer3PLus program. The finished primers and assays were obtained from BioRad or the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences. A T100TM thermal cycler, a QX200TM droplet generator, a PX1TM PCR plate sealer, and a QX200TM droplet reader were obtained from BioRad. The reaction PCR was prepared using ddPCR Supermix for Probes (BioRad). The restriction enzymes (HaeIII, EcoRI, MspI, XbaI) were added to the reaction mix to improve the distribution of DNA across the droplets.

RNA was isolated from immunoprecipitates and cultures using the RNeasy Microisolation kit (Qiagen). Fluorimetry with Ribogreen (Invitrogen) was used for RNA quantification. For analyses of total RNA levels and inputs for RIP analyses, RNA yields were normalized across samples prior to reverse transcription using Sensifast (Bioline). For RIP assays, an equal proportion of each RIP was used for reverse transcription with Sensifast. ddPCR products were detected using Evagreen or Taqman primer and probe sets (Biorad or Integrated DNA Tech; sequences available on request) and QX200^TM droplet reader (Biorad). In GFP RIP experiment, B domain-BFP expression consistently increased G3BP1-GFP levels in the DRG neurons. So the level of mRNA precipitating with G3BP1-GFP was normalized to the G3BP1-GFP signals from immunoblotting across each sample in each experiment.

DNA was extracted from five 10-µm-thick tissue sections using the Maxwell^® RSC (Promega, Madison, WI 53711-5399, USA) device according to the manufacturer’s instructions. Samples were stored at 4 °C until use. BRAF and NRAS mutations were detected by digital droplet PCR (Biorad, Hercules, CA 94547, USA; Cat. No. 12001037, Cat. No. 12001006). Primers and probes for wild-type and mutated BRAF and NRAS genes were purchased by Biorad (Biorad, Hercules, CA 94547, USA; Cat. No. 12001037, Cat. No. 12001006). The reaction was carried out in a solution containing 10 μL of ddPCR^TM Supermix for probes (no dUTP) (Biorad, Hercules, CA 94547, USA; Cat. No. 1863024), 1 μL of mutations Screening Assay, and 25 ng of DNA. The droplets were generated using a QX200^TM Droplet Generator (BioRad, Hercules, CA 94547, USA). Subsequently, emulsions were transferred onto a 96-well PCR plate (Biorad, Hercules, CA 94547, USA) and submitted to PCR amplification as follows: denaturation for 10 min at 95 °C, 40 cycles of 94°C for 30 s, annealing/extension for 1 min at 55 °C, and one cycle at 98 °C for 10 min. After amplification, droplets were read in the QX200^TM Droplet Reader (BioRad, Hercules, CA 94547, USA). Data analysis was performed using QuantaSoft^TM software.