Transcriptomic profile of a MECHANICALLY ACTIVE 3D GUT-ON-CHIP  exposed to human FECAL MICROBIOME.
Transcriptomic profile of a MECHANICALLY ACTIVE 3D GUT-ON-CHIP  exposed to human FECAL MICROBIOME.
To perform RNA sequencing analysis, epithelial cells from Gut-on-Chip devices (n≥3 for each condition) were collected, at day 9 post treatment, flushing trypsin-EDTA solution (Euroclone) in the channels and incubating the device for 10-20 minutes at 37°C. Collected pellets were centrifuged, resuspended in 350 ml of freezing solution (40 mM Dithiothreitol DTT solution - Merck, in RLT buffer - Qiagen) and stored at -80°C until further processing Base calling was performed in real time during the Novaseq runs by Real-Time Analysis (RTA, Illumina) software RNA was extracted from frozen samples by using the RNeasy Mini Kit (Qiagen cat. no. 74106) and on-column digestion DNA was performed using the RNAse-Free DNase Set (Qiagen cat. no. 79254) following manufacturer instructions. RNA was eluted in 50 ml of RNAse-Free water. Sample quality and quantity were checked by Bioanalyzer and Qubit, respectively. Total RNA was depleted of ribosomal RNA with the Ribo-Zero™ rRNA Removal Kit. Efficiency of ribosomal RNA removal was checked with Bioanalyzer 2100. RNAseq libraries were prepared with the Illumina TruSeq Stranded Total RNA kit following the manufacturer's protocol. Briefly, after the fragmentation of RNA, cDNA was synthesized, end-repaired and 3'-end-adenylated. Following adapter ligation, libraries were amplified by PCR. Amplified libraries were checked on Bioanalyzer 2100 and quantified with PicoGreen reagent.