*4.3. Plagl1 Timepoint Expression qPCR*

Animal experiments for this qPCR and for RNA-Scope were approved by the Iowa State University Institutional Animal Care and Use Committee (Protocol IACUC-18–350) and conformed to the NIH Guidelines for the Care and Use of Laboratory Animals. Placenta tissue was collected from e7.5 and e9.5 timed-pregnant mice, as described previously [13], for a total of three biological replicates per timepoint. For e7.5, 12 placentas were collected per biological replicate and for e9.5, one placenta was collected per biological replicate in 500 ul PBS supplemented with 1X protease inhibitor. RNA was isolated using the Purelink RNA Micro Scale kit (Thermofisher Scientific 12183016) following the recommended protocol for purifying RNA from animal tissue. Isolated RNA concentrations were measured using the Nanodrop lite (Thermofisher Scientific ND-LITE, Waltham, MA, USA) and 200–400 ng of RNA was converted into cDNA for each biological replicate with the Applied Biosystems cDNA Reverse Transcription kit (Thermofisher Scientific 4368814). *Plagl1* expression was quantified by RT-qPCR on the CFX connect real time PCR system (BioRad 1855201) with 6ng of cDNA per biological replicate. *Polr2a* was used for normalization of *Plagl1* expression (∆CT). Primer sequences and efficiencies can be found in Supplemental Table S5.
