209 Methods Study population Patientswith an establisheddiagnosis of IBDwere included at the outpatient clinic of theUniversity Medical Center Groningen (UMCG) based on their participation in the 1000IBD project, for which detailed phenotypic information and multi-omics profiles had been generated.99 Patients included in this study were at least 18 years old and were enrolled from 2003–2019. Diagnosis of IBD was based upon clinical, laboratory, endoscopic and histopathological criteria, with the latter criteria also used to determine the inflammatory status of collected biopsies. Detailed phenotypic data were collected for all patients, including age, sex, BMI (body weight divided by squared height), smoking status, Montreal disease classification, medication usage, history of surgery, clinical disease activity and histological disease activity, and all were assessed at time of sampling. Montreal disease classification was recorded from the closest visit to the outpatient clinic at time of sampling. Clinical disease activity was established using the Harvey-Bradshaw Index (HBI) for patients with CD and the Simple Clinical Colitis Activity Index (SCCAI) for patients with UC. We further included 16 healthy non-IBD controls (n=59 biopsies) who underwent endoscopy because of clinical suspicion of intestinal disease or within the context of colon cancer screening. All participants provided written informed consent prior to sample collection. This study was approved by the Institutional Review Board (IRB) of the UMCG, Groningen, the Netherlands (in Dutch: ‘Medisch Ethische Toetsingscommissie’, METc; IRB nos. 2008/338 and 2016/424) and was conducted according to the principles of the Declaration of Helsinki (2013). Mucosal RNA-sequencing 711 intestinal biopsies from 420 patients with IBD were collected. These were immediately snap-frozen in liquid nitrogen by an endoscopy nurse or research technician present during the endoscopic procedure. Biopsy inflammatory status was assessed based on histological examination by certified pathologists. Biopsies were stored at -80°C until further processing. RNA isolation was performed using the AllPrep DNA/RNA mini kit (Qiagen, reference number: 80204) according to manufacturer’s instructions. Homogenization of intestinal biopsies was performed in RLT lysis buffer including β-mercaptoethanol using the Qiagen Tissue Lyser with stainless steel beads (diameter 5 mm, reference number: 69989). For the first sample batch, sample preparation was executed using the BioScientific NEXTflexTM Rapid Directional RNA-Seq Kit (Perkin-Elmer). Paired-end sequencing of RNA was performed using the Illumina NextSeq500 sequencer (Illumina). For the second sample batch, sample preparation was performed for construction of the Eukaryotic Transcriptome Library (Novogene). Paired-end sequencing of RNA was performed using the Illumina HiSeq PE250 platform. Sequencing was performed in two different batches, which necessitated pseudo-randomization (covering type of IBD diagnosis, biopsy location and disease activity) across plates to mitigate potential batch effects. The batch effects have been taken into account in all the analyses. On average, approximately 25 million reads were generated per sample. Raw read quality was checked using FastQC with default parameters (ref v.0.11.7). Adaptors identified by FastQC were clipped using Cutadapt (ref v1.1) with default settings. Sickle (ref v1.200) was used to trim low-quality ends from the reads (length <25 nucleotides, quality <20). Reads Mucosal host-microbe interactions in IBD
RkJQdWJsaXNoZXIy MjY0ODMw