Cfx96 and cfx384 real time pcr detection systems

Samples were collected in Trizol. Total RNA from hPSC-derived samples was isolated by chloroform phase separation using Phase Lock Gel-Heavy tubes, precipitated with ethanol, and purified using RNeasy Mini Kit (Qiagen) with on-column DNA digestion step. RNA from mouse cells was isolated using Direct-zol microprep kit (Zymo research, R2060). cDNA was generated using the iScript Reverse Transcription Supermix (Bio-Rad) for RT–qPCR and quantitative PCR (qPCR) reactions were performed using SsoFast EvaGreen Supermix (Bio-Rad) according to the manufacturer’s instructions in 96 or 384-well qPCR plates using CFX96 and CFX384 Real-Time PCR Detection systems (Bio-Rad) using 5–10 ng cDNA / reaction. Primers were from Quantitect Primer assays (QUIAGEN) except for the ones in Supplementary Table 4. Results were normalized to the housekeeping genes GAPDH or TBP.

Total RNA from first trimester placental tissue and chorionic villous explants was isolated using the RNeasy mini kit (Qiagen, Hilden, DE). RNA (250 ng) was reverse transcribed to cDNA with SuperScript II Reverse Transcriptase (Life Technologies, Carlsbad, CA, USA) as per manufacturer's guidelines. MMP15 and HLA‐G expression was determined by RT‐qPCR using FAM‐labeled TaqMan gene expression assays (Life Technologies, MMP15: Hs00233997_m1; HLA‐G: Hs00365950_g1), TaqMan universal PCR master mix (Life Technologies) and the CFX96 and CFX384 real‐time PCR detection systems (BioRad Laboratories, Hercules, CA, USA). To test for compensatory changes of MMP15 knockdown by another key MT‐MMP, MMP14, and β‐actin expression were also quantified (Life Technologies, MMP14: Hs01037003_g1; ACTB: Hs01060665_g1). Ct values were automatically generated by the BioRad CFX Manager 3.0 software and relative gene expression was expressed as −ΔCt or calculated by the 2^−ΔΔCt method, with HLA‐G as the reference gene. Fetal sex was determined based on XIST (Life Technologies, Hs01079824_m1) and DDX3Y (Life Technologies, Hs00965254_gH) expression as described elsewhere.³¹