Rna lysis buffer

After two days of co-culture, the flow was stopped, and the GMPS set-up was removed from the incubator. Via the seed port, PBS++ was used to wash the apical channel, and 300 μL RNA lysis buffer (cat no. T2010, New England Biolabs Inc.) was applied to the channel and vigorously pipetted. RNA was purified following the manufacturer’s protocol (cat no. T2010, New England Biolabs Inc.). Oligo(dT)_12–18 (cat no. 18418012, Thermo Fisher) was used to reverse transcribe Caco-2 RNA (C1000 Touch Thermal Cycler, BioRad). The resulting cDNA was cleaned and concentrated (cat no. D40144, Zymo Research) and 1 μL was used for qPCR. An SYBR Green-based qPCR master mix (cat no. M3003, New England Biolabs Inc.) and primers (Table S3.) were used to amplify genes of interest (C1000 Thermal Cycler with CFX96 Real-Time System, BioRad). Primers were made with NCBI Primer Blast.⁷⁵ The target gene, GADD45a, was normalized to GAPDH. Relative expression differences were calculated using the delta-delta Ct method.

After two days of co-culture, the flow was stopped, and the GMPS set-up was removed from the incubator. Via the seed port, PBS++ was used to wash the apical channel, and 300 μL RNA lysis buffer (cat no. T2010, New England Biolabs Inc.) was applied to the channel and vigorously pipetted. RNA was purified following the manufacturer’s protocol (cat no. T2010, New England Biolabs Inc.). Oligo(dT)_12–18 (cat no. 18418012, Thermo Fisher) was used to reverse transcribe Caco-2 RNA (C1000 Touch Thermal Cycler, BioRad). The resulting cDNA was cleaned and concentrated (cat no. D40144, Zymo Research) and 1 μL was used for qPCR. A SYBR Green-based qPCR master mix (cat no. M3003, New England Biolabs Inc.) and primers (Table S3.) were used to amplify genes of interest (C1000 Thermal Cycler with CFX96 Real-Time System, BioRad). Primers were made with NCBI Primer Blast⁷⁶ . The target gene, GADD45a, was normalized to GAPDH. Relative expression differences were calculated using the delta-delta Ct method.