High-throughput screening is one of the pillars of drug development. Unbiased transcriptome profiling is now widely used for a deeper understanding of a drug's mechanisms of action, off target effects, and cytotoxicity. Although currently available high-throughput RNA-Seq (HT RNA-Seq) methods such as PLATE-Seq, DRUG-Seq, and BRB-Seq serve these purposes, the inherent nature of these methods does not allow sample-wise sequencing library quality control. Here, we describe an HTR method called High-throughput CellulAr RNA Sequencing (HiCAR-Seq). HiCAR-Seq was optimized to work directly on cultured cells (as little as 1,000 cells) or 10 ng of total RNA. HiCAR-Seq involves reverse transcription from cultured cells or total RNA using oligo-dT primers followed by the PCR amplification of full-length cDNAs using sample-specific barcode primers in individual plate wells. Amplification of cDNA from every sample can be verified using Bioanalyzer. This step not only reveals cDNA amplification but also provides greater precision for pooling equal concentrations of cDNA from different samples. A single pooled cDNA library is made suitable for sequencing on Illumina sequencers using a tagmentation kit. Because HiCAR-Seq targets a small region at the 3' of the mRNAs, as little as 3 to 4 million reads/sample are enough to infer changes in gene expression in human or mouse cells. We believe that HiCAR-Seq represents a robust and competitive addition to the existing set of transcriptome-based high-throughput screening methods. © 2020 Wiley Periodicals LLC. Basic Protocol 1: cDNA synthesis and barcoding/enrichment PCR Basic Protocol 2: Nextera tagmentation/amplification, quantification, and sequencing.
Curr Protoc Mol Biol (Current protocols in molecular biology)
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