*4.9. siRNA knockdown and qPCR*

HTR-8/SVneo (ATCC® CRL3271™) cells were cultured in RPMI-1640 (ATCC 30-2001) with 5% FBS. Cells were seeded at 15 <sup>×</sup> <sup>10</sup><sup>4</sup> cells/well in a 6-well plate and grown for 48 h before transfecting. Transfections were carried out with the Lipofectamine RNAiMAX Transfection Reagent (Fisher Scientific 13778150) and with *PLAGL1* siRNAs (ThermoFisher Scientific 4392420-s10602, s10603) or a negative control (ThermoFisher Scientific 4390843). We performed a media change 24 h after transfection. RNA was extracted 48 h after transfection using the Qiagen Mini RNA kit and quality was determined

using the Bioanalyzer Total RNA nano analysis kit (Agilent, Santa Clara, CA, USA) and all RNA Integrity Numbers were greater than 8. Concentrations were determined using the Nanodrop and 1000 ng of RNA from eight biological replicates was collected for RNA-seq. 400 ng of RNA was then converted to cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific 4368814). *PLAGL1* knockdown was quantified by Real-Time qPCR. *GAPDH* was used for normalization and percent knockdown of *PLAGL1* was calculated using the ∆∆CT method. Primer sequences and efficiency values can be found in Supplemental Table S5.
