Droplet generator oil

DNA was digested with the restriction enzyme BamH1 (New England Biolabs, Ipswich, MA) for 30 min at 37 °C, diluted 1:5 with PCR-certified water. The digested diluted DNA was mixed with the HTLV-1 tax (or gag) and RPP30 primers and probes and Bio-Rad 2× Supermix, which was then emulsified with droplet generator oil (Bio-Rad, Hercules, CA) using a QX-100 droplet generator according to the manufacturer’s instructions. The droplets were then transferred to a 96-well reaction plate (Eppendorf, Hauppauge, NY) and heat-sealed with pierceable sealing foil sheets (Thermo Fisher Scientific, West Palm Beach, FL). The duplex PCR amplification was performed in this sealed 96-well plate using a GeneAmp 9700 thermocycler (Applied Biosystems, Grand Island, NY) with the following cycling parameters: 10 min at 95 °C, 40 cycles consisting of a 30-s denaturation at 94 °C and a 60-s extension at 59 °C, followed by 10 min at 98 °C and a hold at 12 °C.
Following PCR amplification, the 96-well plate was transferred to a QX100 droplet reader (Bio-Rad, Hercules, CA). Each well was queried for fluorescence to determine the quantity of positive events (droplets), and the results were displayed as dot plots (Fig. 1).

Fshr mRNA levels were quantified using droplet digital PCR (ddPCR). In brief, RNA was isolated by TRIzol (Life Technologies). Reverse transcription was performed using SuperScript III reverse transcriptase (Life Technologies). Isolated RNA was used to perform ddPCR to determine Fshr mRNA levels using FSHR Probe (Life Technologies, Cat. #4331182). Droplets containing the cDNA were generated using a Biorad Droplet generator (QX200) by mixing with droplet generator oil (Cat. #D9161172A), and the formed droplets were amplified using a thermocycler (Applied Biosystems) and analyzed using droplet reader (Biorad).

To determine the expression level of BdUEV1 genes in different development stages and in response to abiotic stress, ddRT-PCR was performed by using a Bio-Rad QX200^TM ddPCR System. All reagents and consumables for the experiments including droplet generator oil, DG8^TM cartridges and gaskets, droplet reader oil, and ddPCR supermix for EvaGreen were purchased from Bio-Rad. Briefly, the Droplet Digital PCR began by partitioning the reaction mix containing EvaGreen Supermix, primers and sample cDNA into aqueous droplets in oil via the QX200 Droplet Generator; after transfer of droplets to a 96-well PCR plate, a thermocycling protocol including 95°C for 5 min; 95°C for 30 s, 40 cycles; 60°C for 60 s, 40 cycles (ramp rate set to 2°C/s); 4°C for 5 min; 90°C for 5 min and 4°C infinite was carried out in a conventional thermal cycler. The PCR plate was then transferred to the QX200 Droplet Reader for automatic reading of samples. QuantaSoft^TM Software was used for data analysis. The primers used to amplify the BdUEV1s are listed in Supplementary Table S3.

miRNA quantitation was performed by droplet digital PCR (ddPCR, QX200, Bio-Rad, Hercules, CA, USA). Briefly, 3 μl of diluted cDNA (1:100) were mixed with LNA™-specific primers (has-miR-335-5p: cat. no: YP02119293, and has-miR-657: cat. no: YP02108736, Qiagen, GmbH, Hilden, Germany), and with ddPCR EvaGreen Supermix (Bio-Rad), which was then emulsified with droplet generator oil (Bio-Rad) using a QX200 droplet generator. The droplets were then transferred to a 96-well reaction plate and heat-sealed with a pierceable sealing foil sheet (PX1, PCR plate sealer, Bio-Rad). The PCR amplification was performed in sealed 96-well plate using a T100 thermal cycler (Bio-Rad) with the following cycling parameters: 10 min at 95 °C, 40 cycles at 94 °C for 30-s and at 58° for 60 s, followed by 10 min at 98 °C and a hold at 4 °C. After PCR amplification, the 96-well plate was transferred to a QX200 droplet reader (Bio-Rad). Each well was queried for fluorescence to determine the quantity of positive events (droplets), and the results were displayed as dot plots. The miRNA concentration was expressed as copies/ng of extracted RNA.

miRNA quantitation was performed by droplet digital PCR (ddPCR QX200, Bio-Rad, Hercules, CA, US). Briefly, 3 µl of diluted cDNA (1:25) was mixed with LNATM-specific primers (Qiagen GmbH, Hilden, Germany), and ddPCR EvaGreen Supermix (Bio-Rad, Hercules, CA, US), which was then emulsified with droplet generator oil (Bio-Rad, Hercules, CA, US) using a QX200 droplet generator, according to the manufacturer’s instruction. The droplets were then transferred to a 96-well reaction plate and heat-sealed with a pierceable sealing foil sheet (PX1, PCR plate sealer, Bio-Rad, Hercules, CA, US). PCR amplification was performed in sealed 96-well plate using a T100 thermal cycler (Bio-Rad, Hercules, CA, US) as follows: 10 min at 95 °C, 40 cycles at 94 °C for 30-s and at 58° for 60 s, followed by 10 min at 98 °C and a hold at 4 °C. The 96-well plate was then transferred to a QX200 droplet reader (Bio-Rad, Hercules, CA, US). Each well was queried for fluorescence to determine the quantity of positive events (droplets), and the results were displayed as dot plots. The miRNA concentration was expressed as copies/ng of extracted RNA.