Computational protocol: Redifferentiation of expanded human islet β cells by inhibition of ARX

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Protocol publication

[…] Total RNA was extracted using ZR RNA MiniPrep RNA Isolation Kit (Zymo Research, Irvine, CA), and treated with RNase-free DNase I (Thermo Scientific, Waltham, MA). cDNA was produced using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). qPCR was carried out in triplicates using the Universal Probe Library System (Roche Diagnostics, Indianapolis, IN) in 7300 Real-time PCR system (Applied Biosystems). Results were normalized to TATA-box-binding protein (TBP) or Ribosomal protein large P0 (RPLP0) transcripts. These genes were selected as normalization controls since their detection threshold occurred at the same cycle in all the samples studied. Data analysis was performed with qbase+ software (Biogazelle, Zwijnaarde, Belgium). lists primer sequences. All reactions were performed with annealing at 60 °C. For undetectable transcripts, the cycle number was set to 40 for comparisons. [...] Total RNA was extracted using the ZR RNA MiniPrep RNA Isolation Kit (Zymo Research), and treated with RNase-free DNase I (Thermo Scientific). cDNA libraries were constructed following TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA) for next-generation sequencing. Briefly, 400 ng total RNA was used as starting material and PolyA-selected mRNAs were purified, size-fractioned, and subsequently transcribed into single-stranded cDNA by random hexamer priming. Following second-strand synthesis, double-stranded cDNAs were blunt-end fragmented and indexed using adapter ligation, after which they were amplified and sequenced according to manufacturer’s protocol (Illumina). Libraries were pooled and sequenced on one 50-bp single-read HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina) lane with MiSeq Reagent Kit v3 (Illumina). Standard quality checks for material degradation (Bioanalyzer, Agilent Technologies, Santa Clara, CA) and concentration (Qubit, Life Technologies) were done before and after library construction, ensuring that samples were suitable for sequencing. RNA-seq reads were mapped to the human genome using TopHat2. All samples had more than 20M uniquely mapped reads. Gene expression levels were calculated using HTseq-count utility using Genecode gene annotations (v19). Only genes that were readily detected in the dataset (covered by at least 20 reads; 14,647 genes) were included in subsequent analyses. Quantile normalization was used to normalize expression levels across samples. False discovery rate (FDR) was estimated by 1,000 random gene permutations of the data. Cluster analysis was performed using the CLICK algorithm implemented in the EXPANDER package for gene expression analysis. Heatmaps were created using the gplots R package. Functional enrichment analysis was carried out using DAVID. The set of all genes expressed in the dataset was used as the background set in this analysis. The data was deposited in the GEO database (accession number GSE73433). […]

Pipeline specifications

Software tools qbase+, TopHat, HTSeq, EXPANDER, gplots
Applications RNA-seq analysis, qPCR
Organisms Homo sapiens
Diseases Carcinoma, Renal Cell, Diabetes Mellitus