Ribozol me reagent

The vaginal tract was collected at estrus or at 0.5 dpc for RT-qPCR analysis. Vaginal tissue samples were snap-frozen on dry ice upon removal and stored at −80 °C until use. Tissue samples were homogenized and total RNA was extracted using the RiboZol ME Reagent (Amresco, Solon, OH) according to the manufacturer’s directions. RNA quality and quantity was determined using a NanoDrop 1000 Spectrophotometer (Thermo Scienific). Total RNA (1 μg) was reverse-transcribed using the qScript cDNA SuperMix kit according to the manufacturer’s protocol (Beverly, MA). The cDNA products were diluted 1:5 in nuclease-free H₂O. Diluted cDNA (1 μL) was used as a template for the RT-qPCR reaction using the PerfeCTa SYBR Green FastMix (QuantaBio) according to the manufacturer’s instructions. PCR reactions were run and raw data was recorded on a 7500 Fast Real-Time PCR System (Applied Biosystems, ThermoFisher Scientific). Expression values in vaginal samples were calculated as fold change and normalized to eukaryotic elongation factor 2 (Eef2) expression, relative to the Esr1^f/f. The relative expression of the genes was determined with an n = 3 mice/genotype, each of which were measured in triplicate. Primer sequences for the genes analyzed are listed in Supplementary Table S1.

RNA was extracted from the uteri or human cell lines using RiboZol ME Reagent (Amresco, Solon, OH) according to the manufacturer’s protocol. A total of 1 μg RNA was used as a template for the reverse transcriptase reaction according to the manufacturer’s protocol (qScript cDNA SuperMix, QuantaBio, Beverly, MA). The cDNA products were diluted at 1:10 in nuclease-free H₂O. Diluted cDNA (1 μL) was used as a template for the PCR reaction (PerfeCTa SYBR Green FastMix, QuantaBio) according to the manufacturer’s protocol. PCR reactions were run and raw data were recorded in a 7500 Fast Real-Time PCR System (Applied Biosystems, ThermoFisher). Expression values in uterine samples were calculated as fold change normalized to ribosomal protein L7 (Rpl7) expression, relative to the vehicle or Esr1^f/f. Expression of human KLKs, SPINKs, and ESR1 was calculated relative to RPL5 expression. The primer sequences used in the experiments are listed in S2 Table.