title: Illumina NovaSeq 6000 paired end sequencing; Tissue-dependent transcriptional and bacterial associations in primary sclerosing cholangitis-associated inflammatory bowel disease
design: Tissue-dependent transcriptional and bacterial associations in primary sclerosing cholangitis-associated inflammatory bowel disease
library_construction_protocol: Biopsies were obtained from ileum, caecum and rectum from patients with PSC/UC and patients with UC alone, undergoing colonoscopy for surveillance or disease activity and extension assessment, and non-inflammatory controls undergoing colonoscopy for colorectal cancer screening or chronic abdominal pain/diarrhoea with a macroscopically and microscopically normal colon. Biopsies were collected in RNALaterTM TissueProtect (Qiagen) and stored according to manufacturer guidelines.  On the day of DNA/RNA extraction, samples were thawed at room temperature, RNALater was removed and RLT Buffer (Qiagen) with β-mercaptoethanol (Sigma) was added to each sample and samples were transferred to Soft Tissue Homogenizing CK14 Kit vials (Stretton Scientific). Samples were processed using Precellys 24 (Bertin Instruments) homogeniser (40s at 6500 RPM). 350uL of 70% ethanol was added to homogenised tissue lysates and lysates were transferred to QIAshredder column (Qiagen cat 79656) and subsequently to transferred to AllPrep DNA/RNA MicroKit (Qiagen). An on-column DNase digestion was performed for each sample using an RNAse Free DNAse Kit (Qiagen). Samples were eluted from columns in 30uL RNAse free; DNAse free H2O and RNA concentrations and 260/280 ratios were determined on a NanoDrop 1000 (Thermo Fisher).  Selection of PolyA+ mRNA, generation of double stranded cDNA and library construction were performed using NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760L) with Illumina adapters and barcode tags.