Superscript 3 first

According to the manufacturer’s instructions, total RNA was extracted from approximately 50 mg of colon tissue using the TRIzol reagent, and its quantity was determined using spectrophotometry at 260 nm. The Superscript III first-strand synthesis method was used to generate complementary DNA (cDNA) from 2.5 μg of total RNA (Invitrogen, Carlsbad, CA, USA). QPCR was performed using SYBR Green reagents on a C1000 Touch PCR Thermal Cycler (BIO-RAD Laboratories, Shanghai, China). The primer sequences are listed in Supplementary File S1. mRNA levels were calculated using the 2^−ΔΔCT method and normalized to GAPDH levels.

RNA was extracted from embryos or sorted cells using the Total RNA extraction Kit I (QIAGEN, R6834-02) and subjected to cDNA synthesis using SuperScript III First (Invitrogen, 18080-400). Real-time quantitative PCR was performed using the Fast SYBR Green Master Mix (Applied Biosystems, 4335612) in a QuantStudio 6 Flex Real-Time PCR system or a StepOnePlus Real-Time PCR system following the standard protocols using primers in table S1. Relative gene expression was calculated by comparative Ct method and normalized to Gapdh expression.

According to the manufacturer’s instructions, total RNA was extracted from approximately 50 mg of colon tissue using the TRIzol reagent, and its quantity was determined using spectrophotometry at 260 nm. The Superscript III first-strand synthesis method was used to generate complementary DNA (cDNA) from 2.5 μg of total RNA (Invitrogen, Carlsbad, CA, USA). QPCR was performed using SYBR Green reagents on a C1000 Touch PCR Thermal Cycler (BIO-RAD Laboratories, Shanghai, China). The primer sequences are listed in Supplementary File S1. mRNA levels were calculated using the 2^−ΔΔCT method and normalized to GAPDH levels.