MAC-Seq: Coupling Low-Cost, High-Throughput RNA-Seq with Image-Based Phenotypic Screening in 2D and 3D Cell Models
Journal Title
In: Jenkins, B.J. (ed) Inflammation and Cancer. Methods in Molecular Biology, vol 2691.
Publication Type
Book section
Abstract
Transcriptomic profiling has fundamentally influenced our understanding of cancer pathophysiology and response to therapeutic intervention and has become a relatively routine approach. However, standard protocols are usually low-throughput, single-plex assays and costs are still quite prohibitive. With the evolving complexity of in vitro cell model systems, there is a need for resource-efficient high-throughput approaches that can support detailed time-course analytics, accommodate limited sample availability, and provide the capacity to correlate phenotype to genotype at scale. MAC-seq (multiplexed analysis of cells) is a low-cost, ultrahigh-throughput RNA-seq workflow in plate format to measure cell perturbations and is compatible with high-throughput imaging. Here we describe the steps to perform MAC-seq in 384-well format and apply it to 2D and 3D cell cultures. On average, our experimental conditions identified over ten thousand expressed genes per well when sequenced to a depth of one million reads. We discuss technical aspects, make suggestions on experimental design, and document critical operational procedures. Our protocol highlights the potential to couple MAC-seq with high-throughput screening applications including cell phenotyping using high-content cell imaging.
Publisher
Humana, New York, NY
Keywords
RNA-Seq/methods; *High-Throughput Nucleotide Sequencing/methods; *Gene Expression Profiling/methods; Phenotype; High-Throughput Screening Assays/methods; Sequence Analysis, RNA/methods; 3D cell models; High-content imaging; High-throughput screening; Multiplexing; RNA-seq
Department(s)
Laboratory Research
PubMed ID
37355554
Terms of Use/Rights Notice
Refer to copyright notice on published article.


Creation Date: 2023-10-04 03:42:06
Last Modified: 2024-07-09 05:49:48

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