Dg8 cartridge

DDOST 558C>U RNA-editing assays were performed as described previously with assistance from the MSKCC Integrated Genomics Operation^{28 (link)}. Total RNA was extracted using the RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions. After extraction, the RNA was reverse-transcribed using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). cDNA (20 ng) along with primers purchased from Bio-Rad (10031279 and 10031276) for the target DDOST^558C>U amplification were mixed in PCR reactions in a total volume of 25 μl. Then, 20 μl of the reactions were mixed with 70 μl of Droplet Generation Oil for Probes (Bio-Rad) and loaded into a DG8 cartridge (Bio-Rad). A QX200 Droplet Generator (Bio-Rad) was used to make the droplets, which were transferred to a 96-well plate and the following PCR reaction was then run: 5 min at 95 °C; 40 cycles of 94 °C for 30s and 53 °C for 1 min; and finally 98 °C for 10 min. The QX200 Droplet Reader (Bio-Rad) was then used to analyse the droplets for fluorescence measurement of the fluorescein amidite (FAM) and hexachloro-fluorescein (HEX) probes. The data were analysed using the QuantaSoft analysis software (Bio-Rad) and gating was performed on the basis of positive and negative DNA oligonucleotide controls.

Total RNA was isolated from frozen skeletal muscle with the QIAGEN RNeasy Fibrous Tissue Kit and reverse transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad). The final concentration of digested total DNA was adjusted to fall within the linear range for the Poisson calculation for the expected number of droplets in the digital PCR. Each sample was partitioned into 15,000 droplets on a DG8 cartridge (Bio-Rad) and each droplet was amplified by PCR using the following protocol: initial denaturation step at 95°C for 10 min, 40 cycles of 94°C for 30 s, and 60°C for 60 s, followed by 98°C for 10 min. Primer-probe sets to assess specific full-length or skipped Dmd exon 23 mRNAs have been previously described.²⁵ For amplification of exons from 22 to 24; the percentage of exon skipping was expressed as the total exon 23 skipped transcript as a percentage of total (skipped + unskipped) Dmd transcript. Samples were loaded onto the QX200 droplet reader, and ddPCR data were analyzed with QuantaSoft analysis software.⁶³ (link) The target concentration in each sample was expressed as copies per ng.

Genomic DNA of N. benthamiana and Arabidopsis plants was extracted with cetyl trimethyl ammonium bromide (CTAB) buffer. PPR was used as the internal control for the qPCR assays.
A QX200 droplet digital PCR system (Bio-Rad) was used for droplet digital PCR. Genomic DNA samples of 2 μl (20 ng/μl) were added to 20-μl reaction mixes of BioRad QX200 EvaGreen supermix. The primers were present at final concentrations of 100 nM. The reaction mixes were briefly vortexed, avoiding the formation of bubbles. Each 20 μl reaction mix was loaded into a cell of a BioRad DG8 cartridge followed by addition of a 70-μl droplet generation oil. The cartridge was then placed into the droplet generator for droplet generation. Droplets were transferred to a 96-well PCR plate, heat-sealed with a pierceable foil seal, and PCR amplified. Amplification was conducted under the following standard cycling conditions: 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s; 60 °C for 60 s, 4 °C for 5 min, and 90 °C for 5 min, after which the plate was held at 4 °C; ramp rate 2 °C/s. Following PCR, the plate was put onto the QX200 Droplet Digital reader. Data were collected with Quantasoft™ Software.

Next, CFP10 and Rv1768 DNA copy numbers were quantified in the same samples using the QX100™ Droplet Digital™ PCR system (Bio-Rad, CA, USA). The 20 μl ddPCR mixture comprised of 10 μl of 2 × ddPCR Supermix (Bio-Rad, CA, USA), 1.0 μl of 10 μM sense and antisense primer, 0.5 μl of 10 μM probe, 2 μl of 100 ng/μl whole-blood genomic DNA or plasmid DNA, and RNase/DNase-free water to a final volume of 20 μl. The mixture was placed into the DG8 cartridge with 70 μl of droplet generation oil (Bio-Rad, CA, USA), and the droplets were formed in the droplet generator (Bio-Rad, CA, USA). Subsequently, the droplets were transferred to a 96-well PCR plate (Eppendorf, Hamburg, Germany), and PCR amplification was performed on a S1000 thermalcycler (Bio-Rad, CA, USA) using the following parameters: 95 °C for 5 min, followed by 40 cycles at 95 °C for 30 s and 60 °C for 60 s, with a final hold for 10 min at 98 °C. After amplification, the plate was loaded on the droplet reader (Bio-Rad, CA, USA), and the droplets from each well of the plate were automatically read at a rate of 32-wells per hour. The ddPCR data were analyzed using QuantaSoft analysis software (Bio-Rad, CA, USA), and the quantification of the target genes is presented as the number of copies per μl in PCR mixture. The recombinant plasmids were used as templates to draw the standard curves.

MtDNA deletions were measured by droplet digital PCR as described previously [63 (link)]. Briefly, total genomic DNA was isolated by phenol/chloroform extraction from previously flash frozen muscle. Ten micrograms were restriction digested with TaqI enzyme and extracted again by phenol/chloroform extraction. Droplet PCR reaction mixture and cycling conditions are described elsewhere [63 (link)]. Reaction droplets were made by applying 20 μL of each reaction mixture to a droplet generator DG8 cartridge (Bio-Rad) for use in the QX100 Droplet Generator (Bio-Rad). The thermally cycled droplets were analyzed by flow cytometry in a QX100TM Droplet DigitalTM Reader (Bio-Rad) for fluorescence analysis and quantification of mutation frequencies. The number of target molecules per droplet was calculated automatically by the QuantaSoft software (Bio-Rad) using Poisson statistics as described elsewhere [63 (link)].

The ddPCR reaction was performed in a QX200 Droplet Digital PCR System (Bio-Rad Laboratories, CA) according to the manufacturer’s instruction [11 (link)]. Each test was prepared in a 20 μl volume of the reaction mixture, which comprised 10 μL of 2× QX200™ ddPCR™ EvaGreen® Supermix (no dUTP; Bio-Rad), forward and reverse primers and 4 μL of DNA templates. For microdroplets generation, 20ul mixture and 70 ul droplet generation oil were added to the DG8™ cartridge (Bio-Rad), then loaded into a QX200 Droplet Generator (Bio-Rad). Next, microdroplets were transferred into 96-well PCR plate and heat-sealed with foil to prevent aerosol pollution. Then the PCR was performed on a Bulk PCR Thermal Cycler using the following conditions: Pre-denature for 1 cycle at 95 °C for 10 min; denature for 45 cycles at 95 °C for 15 s; anneal and extend for 45 cycles at 60 °C for 1 min. Finally, the fluorescence signal in each plate was analyzed by a QX200 Droplet Reader and QuantaSoft™ Version 1.7.4 [10 (link), 25 (link)]. Each reaction adopted negative control and was performed in duplicate.