DOI: 10.5061/dryad.rfj6q57dw
title: RNA-Seq and proteomics of Crohn's disease: Terminal ileum of inflamed and non inflamed paired tissue biopsy

description: The cause of chronic inflammation manifestations such as inflammatory
 bowel disease (IBD) is still not yet fully understood. Evidence however
 points to the convergence of environmental factors, genetic factors,
 intestinal microbiota and the immune response at the intestinal epithelial
 surface that consequently leads to a breakdown of barrier function in IBD.
 An increasing number of theories implicate a dysfunctional intestinal
 epithelial surface in the pathogenesis of IBD and progression to
 complicated disease, which contributes significantly to the IBD burden. As
 yet, the significance of the intestinal epithelial surface in IBD
 manifestation and progression has not been explored using a systems
 biology approach, that compares the mucosa of healthy patients to those
 with IBD. In the context of dyregulation of the epithelial barrier, there
 is a scarcity of publications that explore the contribution of proteins-
 the effectors of the cells and RNA message in the pathways affected by CD
 and in particular the localization of inflammatory response. We aim to
 evaluate the functional pathways of chronic inflammation at the intestinal
 epithelial surface using a combination of transcriptomic and proteomic
 analyses on intestinal epithelial tissue samples from paired inflamed and
 non-inflammed Crohn’s disease (CD) and healthy control subjects.
 Concordance of several biological pathways from both data sets was found
 to be altered in CD patients’ epithelia when compared to healthy controls.
 This information could be helpful in identifying novel therapeutic targets
 that aim to restore barrier function at the intestinal epithelial surface
 and to guide therapy.
description: <strong>Collection of
 samples </strong> Pinch biopsies from Crohns disease patients
 undergoing colonoscopy at Concord Repatriation General Hospital were
 collected from inflamed Terminal ileum portion of bowel and the closest
 macroscopically normal mucosa section. Two pinches were collected from
 both the inflamed (I) and non-inflamed (NI) portions of bowel. Pinch
 biopsies were immediately placed in 150μl of RNAlater (ThermoFisher
 Scientific, Melbourne, Australia) and stored at -80°C until use. Bowel was
 graded as either I or NI by the gastroenterologist performing the
 colonoscopy based on the SES-CD. In addition, samples were also collected
 from healthy control patients undergoing colonoscopy for unrelated
 conditions. <strong>Extraction of total
 RNA</strong> 7–20mg of frozen bowel tissue was added to a
 chilled and washed 2ml Eppendorf tube. Lysis buffer from the Qiagen
 RNAeasy Mini Kit (Melbourne, Victoria, Australia) was added along with 6mg
 of 1.0mm zirconium beads previously washed with IPGphor detergent and
 nitric acid, and the tissue was homogenised for 40 seconds in a
 MiniBeadbeater (Biospec Products, Bartlesville, USA) then rested for 2
 minutes, for 8 cycles carried out in a cold room at 4°C. Tubes were spun
 for 3 minutes at 1500 x g. The lysate was transferred to a new RNA/DNA
 free 1.5ml microcentrifuge tube. Total RNA was extracted using the Qiagen
 RNAeasy Mini Kit according to the manufacturer’s instructions. RNA was
 eluted with nuclease-free water supplies with the kit. The quality of the
 RNA was checked by testing 260/280 ratio was &gt;1.8, 260/230 ratio
 was between 1.8–2.2 and the RNA integrity number was &gt;8. Patient
 samples consisted of 5 non-inflamed controls and 44 inflamed Crohn's
 disease terminal ileum (TI) tissue samples biopsied from an inflamed and
 non-inflamed region of the TI patient paired.
 <strong>Analysis of
 total RNA and RNA-Seq bioinformatics
 analysis</strong> Analysis of total RNA from 15 samples was
 performed by the Ramaciotti Centre for Genomics using the NovaSeq platform
 to obtain 100 bp paired end reads. Counts were quantified using salmon (v
 0.9.1) and the R package txtimport using the Ensembl
 annotation file GRCh38.87.fa. A total of 17,556 genes remained after
 filtering out lowly expressed genes. Differential expression analysis was
 performed using the edgeR and limma (voom) R packages and the treat
 function to select for genes with a logFC of at least log2(1.2).
 <strong>Analysis of CD
 tissue proteome </strong> Global proteomics was carried out on protein
 extracted from 7–20mg of frozen bowel tissue in lysis buffer (0.1M DTT,
 0.1M CHAPS, 9M Urea, 3mM Tris) with protease inhibitor and 6mg of 1.0mm
 zirconium beads using a cell shearing method. One hundred
 μg of protein was digested
 in a 1:100 ratio of protein to trypsin at room temperature
 overnight.<br> Proteomic Mass spectrometry was carried out
 using a QExactive Plus (Thermo Electron, Bremen, Germany). Briefly, the
 sample, 1.5 μg (2.0 μL from 10μL), was loaded onto a micro C18 pre-column
 (300 μm × 5 mm, Dionex) with
 H<sub>2</sub>O:CH<sub>3</sub>CN (98:2, 0.1% TFA)
 at 10 μL min<sup>-1</sup>. After washing, the pre-column was
 switched (Valco 10 port valve, Dionex) into line with a fritless
 nanocolumn (75 μm i.d × 20 cm) containing reverse phase C18 media (1.9 μm,
 120 A, Dr. Maisch HPLC GmbH). Peptides were eluted using a linear gradient
 of H<sub>2</sub>O:CH<sub>3</sub>CN (98:2, 0.1% formic acid) to H<sub>2</sub>O:CH<sub>3</sub>CN (64:36, 0.1% formic acid) at 250 nL min−1 over 60 min. The QExactive mass spectrometer was run in DDA mode where a high voltage of 2000 V was applied to a low volume union and the column (45 ◦C) positioned 0.5 cm from the heated capillary (275°C). A survey scan 350–1750 m/z was acquired in the Orbitrap (resolution 70,000 at 200 m/z) with an accumulation target of 106 ions, lock mass enabled and up to the 10 most abundant ions (AGC target set to 105, minimum AGC target set to 1.5 × 104) with charge states ≥ +2 and ≤ +6 sequentially isolated and fragmented.