SI organoids were stimulated with ENR (control) or ENR + Hpb-CM medium for 24 h from the start of culture. RNA was extracted using Trizol reagent (Sigma-Aldrich), and quality was checked with the Bioanalyzer RNA 6000 kit. Libraries were prepared using the NEB mRNA stranded library prep Kit (New England Biolabs) and paired-end sequenced on the NovaSeq 6000 at 25 million reads per sample. RNA-seq reads were aligned to the mm10 reference genome using STAR (Dobin et al., 2013 (link)). Read counts were calculated using the strand-specific exonic reads of each gene, and duplicate genes were merged using HOMER (Heinz et al., 2010 (link)). Transcripts per million were used to evaluate the correlation among replicates for quality control. Differential gene expression was calculated from the raw read counts using edgeR (Robinson et al., 2009 (link)). Data sets presented in the manuscript can be found at GEO, accession no. GSE199227 .
Neb mrna stranded library prep kit
Manufactured by New England Biolabs
The NEB mRNA Stranded Library Prep Kit is a laboratory tool designed for the preparation of stranded mRNA libraries for sequencing. The kit provides the necessary reagents and protocols to generate high-quality cDNA libraries from total RNA, while preserving the original orientation of the mRNA molecules.
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2 protocols using neb mrna stranded library prep kit
1
RNA-Seq Analysis of Organoid Responses
2
RNA-seq Data Processing and Analysis
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Extracted RNA was controlled for integrity using the Agilent Bioanalyzer (RNA Analysis), Stranded libraries were prepared using the NEB mRNA stranded library prep Kit (New England Biolabs) and sequenced using HiSeq2500 sequencers (Illumina). Raw reads are clipped for adapter sequence, trimmed for minimum quality (Q30) in 3’ and filtered for minimum length of 32 bp using Trimmomatic.44 (link) Surviving read pairs were aligned to Mus_musculus assembly GRCm38 by the ultrafast universal RNAseq aligner STAR45 (link) using the recommended two passes approach. Aligned RNA Seq reads were assembled into transcripts and their relative abundance was estimated using Cufflinks and Cuffdiff.46 (link) Exploratory analysis was conducted using various functions and packages from R and the Bioconductor project. Differential expression was conducted using both edgeR and DEseq. Terms from the Gene Ontology were tested for enrichment with the GOseq47 (link) R package.
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