Rneasy powerlyzer tissue and cells kit

RNA was extracted from colon biopsy tissue using the RNeasy Powerlyzer Tissue and Cells kit per manufacturer’s protocol (QIAGEN). Briefly, tissues were homogenized with two 45-second cycles at 5.5 m/s in the FastPrep 24, and an on-column treatment with RNase-free DNase was included. RNA was extracted from PBMC using the RNeasy Mini Kit, including QIAshredder and RNase-free DNase treatments (QIAGEN). An RNase inhibitor was added to eluted RNA before storage at −80 °C. RNA was quantified with the Qubit RNA BR assay kit (Thermo Fisher Scientific, Waltham MA, USA), and quality was assessed by spectrophotometry and by a RNA High Sensitivity ScreenTape assay (Agilent, Santa Clara, CA, USA). A buffer-only extraction was included in each tissue or PBMC extraction batch.

Total RNA was extracted from intestinal tissue using the RNeasy PowerLyzer Tissue and Cells kit from Qiagen (Germantown, MD, USA), according to the manufacturer’s instructions. Then, 2 µg RNA was reverse-transcribed to complementary DNA (cDNA) using the OneStep RT-PCR kit (Life Technologies, Grand Island, NY, USA) in a Bio-Rad C1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA). Quantitative PCR was performed using QuantiFast SYBR Green PCR kit (Qiagen, Germantown, MD, USA) in a StepOne Real-Time PCR System (Thermo Fischer Scientific, Waltham, MA, USA). GAPDH was used as control for normalization. Primers were obtained from Integrated DNA Technologies (Coralville, IA, USA), and sequences are as follows: Sesn2, forward: 5′-GAGCTGGAGAAGTCAGAAAG-3′, reverse: 3′-GGTCCTCCACAAAGCATAG-5′.