title: Pulmon_Non-tumor
design: Thirty-eight CRC and nineteen NSCLC samples, and their paired non-tumor tissues, were obtained from patients who had undergone potentially curative surgery at San Carlos Hospital in Madrid, Spain, along the last 4 years. Mean age of CRC patients (24 males and 14 females) was 71.24  12 (mean  standard deviation). For NSCLC cases (9 males and 10 females), the mean age was 72.79  7.91 (mean  standard deviation). After surgical resec-tion, all tissue samples were instantly frozen in liquid nitrogen and stored until processed at -80C in the Biobank of the Health Research Institute of San Carlos Hospital (IdISSC) in Madrid, using Tissue-Tek OCT as a freezing medium.Prior to DNA extraction, tumor and non-tumor tissue samples from CRC and NSCLC patients were washed to remove the Tissue-Tek OCT, by the addition of 1ml PBS 1x and subsequent centrifugation at 3000 rpm and room temperature for 15 minutes. Next, total DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen, Hilden, Ger-many) following the manufacturers protocol. After the extraction, DNA concentration was measured with the Invitrogen Qubit 3 Fluorometer and the dsDNA HS (high sensitiv-ity) Assay (Thermo Fisher Scientific, Waltham, MA, USA).  Microbiota analysis consisted in the amplification and sequencing of the bacterial 16S ribosomal RNA (rRNA) gene, using the Ion Torrent technology and reagents from Life Technologies (a division of Thermo Fisher Scientific, Waltham, MA, USA). The protocol followed for library preparation and sequencing has been described previously [8]. Shortly, DNA from each sample was submitted to 2 PCR reactions in parallel, each one starting from 5 ng of input DNA, and using the reagents from the Ion 16S Metagenomics Kit. Re-actions were set to 25 cycles, with the conditions mentioned in the manufacturers proto-col. Two different primer sets were used to amplify 7 hypervariable regions of the gene (set 1, regions V2, V4 and V8; and set 2, regions V3, V6-7 and V9). The obtained amplicons from both reactions were quantified using the Qubit, combined equimolarly for each sample and purified with the CleanPCR reagent (CleanNA, Waddinxveen, The Netherlands). Next, barcoded libraries were prepared with the reagents from the Ion Plus Fragment Library Kit and the Ion Xpress Barcode Adapters 1-96 Kit. Resulting final libraries were quantified with the Qubit, diluted to 22 pM and pooled together. An emulsion PCR was then per-formed using the Ion OneTouch 2 System and the Ion 520 & 530 KitOT2, from which Template-positive ion sphere particles (TP-ISPs) were obtained. Finally, these TP-ISPs were collected, washed, enriched using the Ion OneTouchES Instrument OT2, and loaded onto an Ion 530 Chip. Sequencing was performed with the Ion S5 System.
identifiers.namespace: SUB14956228
identifiers.id: CP18-N