Trizol solution

Diclofenac sodium, ibuprofen, paracetamol, isopropyl alcohol, PEG400, and 75% alcohol were obtained from Sigma-Aldrich (St. Louis, MO, USA). Diclofenac sodium salt was solubilized in PEG400. TRIzol solution and a cDNA synthesis kit were purchased from ZYMO RESEARCH (Irvine, CA, USA). TB Green^® Fast qPCR Mix was purchased from Takara Bio (Kusatsu, Japan). The oligonucleotides, for PCR reaction, were bought from Integrated DNA technologies (Coralville, IA, USA).

NanoVelcro Chips were used for isolating the single and clustered cTBs from pregnant women. To extract RNA from the single and clustered cTBs captured on NanoVelcro Chips, we performed on-chip lysis of cells by introducing 600 µL of TRIzol solution (Zymo Research, USA) and 600 µL of anhydrous ethanol (Sigma-Aldrich) sequentially through the NanoVelcro Chips. The effluent solution was collected in a 2.0 mL RNase-free Eppendorf tube at the same time. Then, RNA was purified using a Direct-zol™RNA MicroPrep Kit (Zymo Research). cDNA was synthesized using a Thermo Scientific Maxima H Minus Reverse Transcriptase Kit according to the manufacturer’s instructions. cDNA was then tested for the trophoblast-specific gene using duplex ddPCR in each tube with two fluorescence filters (i.e., FAM and VIC). RT-ddPCR experiments were performed using the same protocol as that used for the placenta tissues.