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Transcriptome Analysis of Plasmodium CSP stable expressing or control A549 cells

ABSTRACT: To investigate the potential effect of overexpression of Plasmodium yoelii cirumsporozoite protein on A549 cells. Control and CSP stable expression A549 cells were constructed by infected with pLenti vector and pLenti-CSP plasmids and cultured with HBSS for 24h. Total RNA from Control and CSP stable expression A549 cells was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer’s protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The samples with RNA Integrity Number (RIN) ≥ 7 were subjected to the subsequent analysis. The libraries were constructed using TruSeq Stranded mRNA LTSample Prep Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Then these libraries were sequenced on the Illumina sequencing platform (illumina novaseq6000) and 125bp/150bp paired-end reads were generated.

ORGANISM(S): Homo sapiens

PROVIDER: GSE189311 | GEO | 2022/12/31

REPOSITORIES: GEO

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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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Transcriptome Analysis of Plasmodium CSP stable expressing or control A549 cells

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