Reverse transcription reagent

To avoid mixing the 2 populations of cells during the RNA isolation, the Matrigel layer containing IECs was gently delayered using a spatula, ensuring physical separation from fibroblasts on the bottom of the plate (Fig. S6), and transferred to microcentrifuge tubes. To recover IECs embedded in Matrigel for further analysis, the Matrigel layer was digested with a Cell Recovery Solution (cat# 354253, Corning, NY, USA) and then centrifuged at 1,000 × g at 4 °C for 10 min. The cell pellets were resuspended in TRIZOL reagent (cat# 15596018, Ambion, CA, USA) for RNA isolation. RNA was converted to cDNA using reverse transcription reagents (cat# KMM-101, TOYOBO, Japan), according to the manufacturer’s instructions. qPCR was performed using the SYBR Green Real time PCR Master Mix Kit (cat# 4472918, TOYOBO, Osaka, Japan) with the primers listed in Table S1. The reaction was performed using an ABI StepOne plus real-time PCR machine (Applied Biosystems QuantStudio 5, CA, USA) at the Soonchunhyang Biomedical Research Core Facility of the KBSI. All experiments were performed with at least 3 biological replicates for each group, and the expression levels of genes of interest were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The ΔCt values were determined as follows: Ct^target − Ct^GAPDH, and relative fold changes were calculated using the 2^−ΔΔCt method [33 (link)].

Total RNA was extracted using TRIzol reagent (Cat #15596018, Invitrogen) from the collected middle part of the colon. RNA was converted to cDNA using reverse transcription reagents (Cat #FSQ-201, Toyobo). PCR was performed using SYBR Green Real time PCR Master Mix Kit (Toyobo) with specific primers for target genes (Table S12). The reaction was performed with QuantStudio5 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The expression levels of target genes were calculated by comparing the relative expression levels after normalization to β-actin.

Total RNAs were extracted from hPSCs, mesendodermal cells, IM cells, MDLCs and endometrium organoids using Trizol Reagent (Toyobo). The concentration of total RNA was measured by NanoDrop (Thermo Fisher Scientific). Each sample was reverse-transcribed using the Reverse transcription reagent (Toyobo) to produce cDNA. The cDNA was quantified by real-time qPCR using SYBR Green qPCR Master Mix (Takara) and CFX96 Real-Time PCR Detection System (Bio-Rad). Quantification of the samples was performed according to the threshold cycle using the ΔΔCt method. These experiments were repeated three times. The primers are listed in Supplementary Table 1.

Fresh overnight cultures of G. vaginalis in NYCIII (growth medium) and sBHI (biofilm medium) were used to assess the bacterial cells in planktonic and biofilm stages. Total RNA was extracted using the TRIzol Max Bacterial RNA Isolation Kit (Thermo Scientific) according to the manufacturer’s instructions. Reverse transcription reagent (Toyobo) was used to reverse transcribe total RNA. The expression levels of sialidase, vaginolysin, bacitracin transport ATP-binding protein (BcrA), and multidrug resistance ABC transporter (ABC transporter) gene were analyzed by quantitative real-time PCR (qRT-PCR) using SYBR Green PCR Master Mix (Toyobo) and an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster, CA, United States). The cycling steps were: 5 min at 95°C, followed by 40 cycles of 15 s at 95°C and 60 s at 56°C. The primers used for qRT-PCR are listed in Supplementary Table 1.