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Amplicon libraries of ITS1 genes

ABSTRACT: DNA was extracted within 24 hour using the MoBio PowerSoil DNA Isolation kit (Qiagen, Valencia, CA, USA). Nested PCR was used to amplify the fungal internal transcribed spacer 1 (ITS1) using barcoded-primers. PCR-amplified products were pooled and purified using the AxyPrep DNA Gel Extraction kit (Axygen, Tewksbury, MA, USA) according to the manufacturer’s instructions, then quantified with the dsDNA HS Assay (Invitrogen, Darmstadt, Germany) and used for library construction for Illumina sequencing with the LTP Library Preparation kit (KAPA Biosystems, Boston, MA, USA). Sequencing data were obtained with an Illumina Miseq platform (San Diego, CA, USA) using the 2*300 paired-end protocol.

SUBMITTER: Ting Zhu

PROVIDER: OEX00002156 | NODE | 2020-03-24

REPOSITORIES: NODE

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Project description:Introduction: Inflammatory bowel disease (IBD) is a complex multi-factorial disease for which physiologically relevant in vitro models are lacking. Existing models are often a compromise between biological relevance and scalability. Here, we integrated intestinal epithelial cells (IEC) derived from human intestinal organoids from 3 different donors in a gut-on-a-chip platform to model the human intestine and key aspects of IBD. The goal of our transcriptomics experiment was to compare microfluidic-grown IEC to donor-paired HIO cultures in standard Matrigel droplets used for gut-on-a-chip seeding. Methods: For the microfluidic gut-on-a-chip, IEC cells were seeded in the top channel of a 3-lane OrganoPlate on top of a Collagen I gel embedded in the middle channel. Cells were kept in HISC medium devoid of A83-01 for 2 days, before medium was switched back to HISC medium. 6 days after seeding, cells were triggered with LPS 100ng/mL, IFN-γ 20ng/mL for 24 hours. RNA was isolated with TRI-reagent followed by a phenol-chloroform extraction. For the static Matrigel-grown HIO, RNA was isolated of confluent cultures with TRI-reagent followed by a phenol-chloroform extraction. Library preparations, sequencing reactions were conducted at GENEWIZ, LLC. (South Plainfield, NJ, USA). RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were multiplexed and clustered on one lane of a flowcell and loaded on the Illumina HiSeq instrument according to manufacturer’s instructions. The samples were sequenced using a HiSeq 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification.

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Amplicon libraries of ITS1 genes

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