Computational protocol: Huntingtin Is Required for Neural But Not Cardiac/Pancreatic Progenitor Differentiation of Mouse Embryonic Stem Cells In vitro

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Protocol publication

[…] Total RNA was isolated from three different batches of R1 and HN mESCs collected on different dates using the Direct-zolTM RNA MiniPrep Kit (Zymo Research) according to the manufacturer’s instructions. Bioanalyzer was used to determine RNA concentration and quality. One hundred and thirty nanograms of total RNA was used to construct RNA-Seq libraries using Illumina TruSeq RNA Library Preparation Kit v2 and 15 cycles of PCR amplification. Libraries were run on an Illumina HiSeq 2500 instrument using a paired end 50 protocol, rapid run flow cell.Sequencing results were demultiplexed and converted to FASTQ format using Illumina bcl2fastq software. Reads were aligned to the mouse genome (build mm10/GRCm38) using the splice-aware STAR aligner (). PCR duplicates were removed using the Picard toolkit. HTSeq package () was utilized to generate counts for each gene based on how many aligned reads overlap its exons. These counts were then normalized and used to test for differential expression using negative binomial generalized linear models implemented by the DESeq2 R package (). Additionally, differential exon usage analysis was performed using the DEXSeq R package (). The RNA-seq data has been deposited to the NCBI GEO database with an accession number GSE92905. […]

Pipeline specifications

Software tools BCL2FASTQ Conversion Software, STAR, Picard, HTSeq, DESeq2, DEXSeq
Application RNA-seq analysis
Organisms Mus musculus, Homo sapiens
Diseases Huntington Disease