Computational protocol: Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation[S]

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Protocol publication

[…] Sandhoff disease and HEXB-corrected cerebral organoids were harvested at weeks 8 and 10 of culture and RNA was extracted (four samples at each time point, each consisting of four to six organoids) using the RNeasy Mini kit (Qiagen, Hilden, Germany). RNA was quantified using the Agilent RNA 6000 Nano kit (Agilent Technologies, Santa Clara, CA) on a BioAnalyzer 2100 (Agilent Technologies). RNA (1 μg, RIN >8) was used to prepare RNA-sequencing (RNA-Seq) libraries with the TruSeq Stranded mRNA Library Prep kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. Library DNA concentrations were measured using the Quant-iT PicoGreen dsDNA assay kit (Thermo Fisher Scientific). All samples were normalized according to concentration and pooled. Single-end 50 bp sequencing was performed on an Illumina HiSeq 2500. Reads were mapped to the human hg19 reference genome using the ELAND aligner (Illumina). Reads per kilobase of transcript per million (RPKM) values were determined using the Genomatix Software Suite (Genomatix, Munich, Germany).Whole transcriptomes from Sandhoff disease and HEXB-corrected organoids at weeks 8 and 10 of culture were compared with RNA-Seq gene expression data from 16 normal human dorsolateral prefrontal cortex specimens at the fetal and infancy developmental stages (obtained from the Allen Brain Atlas; (). Pearson correlation analysis was computed between the gene expression levels, quantified as RPKM, of the four Sandhoff disease and four HEXB-corrected samples for each time point and the values for the 16 control human dorsolateral prefrontal cortex samples (, ). The National Center for Biotechnology Information Gene Expression Omnibus (GEO) accession number for the RNA-Seq data is GSE106311.Genes that were expressed at levels RPKM >3 in both cerebral organoids and brain samples were included for Pearson correlation analysis. Heat maps were generated using Prism 7 software (GraphPad, La Jolla, CA).Differential gene expression analysis was performed by the DESeq2 algorithm using Genomatix Software Suite. A threshold for the log2 (fold change) of expression/enrichment level (HEXB-corrected vs. Sandhoff disease) of 1.5 and an adjusted P-value threshold ≤0.05 (Wald test) were used for the analysis.Gene ontology analysis was performed using Genomatix Pathway System (Genomatix Software Suite) through the analysis of the top 100 upregulated and the top 100 downregulated genes for HEXB-corrected organoids compared with Sandhoff disease organoids at week 10 of culture. The biological processes category was plotted selecting the top 14 terms ranked by P value. […]

Pipeline specifications

Software tools ELAND, Genomatix Software Suite, DESeq2, GePS
Databases GEO Allen Brain Atlas
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Sandhoff Disease, Lysosomal Storage Diseases, Neurodegenerative Diseases
Chemicals G(M2) Ganglioside