Transcriptional Analysis of Cryptosporidium-Infected Organoids

Organoids derived from two independent donors were microinjected with oocyst or control media. Total RNA was extracted from collected organoids at 24 and 72 hr post-injection using RNeasy Micro Kit (Qiagen) according to manufacturer’s protocol including DNaseI treatment. Quality and quantity of isolated RNA was checked and measured with Bioanalyzer 2100 RNA Nano 6000 chips (Agilent). Libraries were prepared as described previously47 (link) and sequenced on an Illumina NextSeq500 by using 75-bp paired-end sequencing. Paired-end reads from Illumina sequencing were aligned to the human transcriptome genome and C. parvum transcriptome genome (Iowa strain) by BWA48 (link). DeSeq (v1.18.0) was used for read normalization and differential expression analysis. Gene set enrichment analysis (GSEA) was performed using gene lists for type I interferon response and regulation against normalized RNA-seq reads of injected SI and lung organoids using GSEA software v3.0 beta2.

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Heo I., Dutta D., Schaefer D.A., Iakobachvili N., Artegiani B., Sachs N., Boonekamp K.E., Bowden G., Hendrickx A.P., Willems R.J., Peters P.J., Riggs M.W., O’Connor R, & Clevers H. (2018). Modeling Cryptosporidium infection in human small intestinal and lung organoids. Nature microbiology, 3(7), 814-823.

Publication 2018

RNeasy Micro Kit Bioanalyzer 2100 RNA Nano 6000 chips NextSeq500

C parvum Chips Donors Gene Genome Genome human Lung Oocyst Organoids Rna seq Strain Transcriptome Type i interferon

Corresponding Organization : Princess Máxima Center

Other organizations : University of Arizona, Maastricht University, University Medical Center Utrecht

Top 5 similar protocols

Variable analysis

independent variables

Oocyst injection
Control media injection

dependent variables

Gene expression changes in organoids at 24 and 72 hours post-injection

control variables

Organoids derived from two independent donors
Total RNA extraction using RNeasy Micro Kit (Qiagen) with DNaseI treatment
RNA quality and quantity checked with Bioanalyzer 2100 RNA Nano 6000 chips (Agilent)
Library preparation as described previously
Sequencing on Illumina NextSeq500 using 75-bp paired-end sequencing
Alignment of paired-end reads to human transcriptome genome and C. parvum transcriptome genome (Iowa strain) using BWA
Read normalization and differential expression analysis using DESeq (v1.18.0)
Gene set enrichment analysis (GSEA) using gene lists for type I interferon response and regulation

controls

Control media injection

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