*4.6. GDM Placenta qPCR*

All animal procedures were approved by the Baylor College of Medicine institutional animal care and use committee and performed in accordance with NIH Guide for the Care and Use of Laboratory Animals. Seven week old C57BL/6J female mice were placed on either a 10% kcal/fat, 0% kcal/sucrose control diet (Ctrl) or a 45% kcal/fat, 17% kcal/sucrose diet (GDM) one week prior to and throughout pregnancy to induce GDM like symptoms as previously described [45]. All females were placed with a proven breeder male for one night and then examined for copulatory plugs in the morning. Plug positive females were considered pregnant and morning of plug positive was designated as day 0.5 of pregnancy. At embryonic day 17.5, dams were euthanized. For GDM mice, 10 placentas were dissected from 5 dams. For control mice, 11 placentas were dissected from six dams. Placenta were individually flash frozen. Flash frozen placenta were thawed overnight in RNA*later*-ICE and RNA extracted using PureLink RNA Mini Kit. Quality of RNA was checked on a 1% Agarose gel. RNA was converted to cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher Scientific 4368814). Primers used for *Plagl1* and *Polr2a* (mouse) can be found in Supplemental Table S5.

Sex of the placenta was determined as previously described [103] using PCR to amplify either *Rbm31x* and *Rbm31y*. Samples were then run on a 1% agarose gel to determine sex (Supplemental Table S3).
