Abstract
RNA sequencing (RNA-seq) has become a popular method for profiling gene expression. Among many applications, one common purpose is to identify differentially expressed genes and pathways in different biological or pathological conditions. This protocol provides detailed procedure for RNA-seq analysis of ~250,000 sorted Drosophila intestinal cells (Chen et al., 2016), in which RNA amplification is not required.
Keywords: RNA-seq, FACS, Drosophila intestine, Progenitor cells, Transcriptome analysis
Background
Transcriptome analysis by RNA-seq has become a popular method for the identification of differentially expressed genes and pathways under different biological or pathological conditions. For samples that yield low mRNA levels, RNA or cDNA amplification was commonly performed before deep-sequencing (Dutta et al., 2015). However, this procedure could potentially omit important candidates that are expressed in low abundance. Here we provide a detailed procedure for RNA-seq analysis of sorted Drosophila gut cells in which RNA amplification is not required.
Materials and Reagents
Equipment
Software
Procedure
Figure 1. Protocol overview. Under RNase-free environment, the midguts with foregut and hindgut portion removed (left panel) are digested with Elastase for 1 h. The dissociated tissues are centrifuged, resuspended in DEPC-PBS, filtered, and then sorted through FACS. About 250,000 sorted cells are collected to harvest total RNA, which is then used for library construction and sequencing with Illumina Hiseq-2500 system. The upper right panel shows a confocal image of midgut epithelium with Esg-GFP expression. The bottom right panel highlights the Esg-GFP+ cell population measured by FACS.
Data analysis
Recipes
Acknowledgments
Sorting of intestinal progenitor cells was performed according to the method described previously (Dutta et al., 2015) with some modifications. This work was supported by National Basic Science 973 grant (2014CB850002) from the Chinese Ministry of Science and Technology.
References
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