16S rRNA sequencing of Sample Sample BEM39, Barrett Esophagus patient, category: BEM
Genomic DNA was isolated from one mucosal biopsy for each condition using High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland) according to manufacturers instructions. 200 l of Tissue Lysis Buffer and 40 l of Proteinase K were added to 25-50 mg of sample material. This suspension was incubated for 3 hours at 55C until tissue was completely digested. Then, 200 l of Binding Buffer were added and incubated for 10 minutes at 70C. Finally, samples were washed with 500 l of Washing Buffer using a column (High pure filter tube) and eluted with 200 l of Elution Buffer. The V3V4 hypervariable regions of the 16S ribosomal RNA (rRNA) gene were amplified according to the 16S Metagenomic Sequencing Library Preparation protocol (Illumina, San Diego, CA, USA) and sequenced on a MiSeq platform (Illumina), in a single 2  300 bp paired-end run. Raw sequencing reads were trimmed down to 250 nt, due to quality.