Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Loontiens et al. BMC Genomics (2019) 20:228 https://doi.org/10.1186/s12864-019-5608-2 METHODOLOGY ARTICLE Open Access Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes Siebe Loontiens1,2, Lisa Depestel1,2, Suzanne Vanhauwaert1,2, Givani Dewyn1,2, Charlotte Gistelinck1,3, Karen Verboom1,2, Wouter Van Loocke1,2, Filip Matthijssens1,2, Andy Willaert1, Jo Vandesompele1,2, Frank Speleman1,2 and Kaat Durinck1,2* Abstract Background: Transgenic zebrafish lines with the expression of a fluorescent reporter under the control of a celltype specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis. Results: We evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results. Conclusions: The presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells. Keywords: RNA isolation, FACS sorting, Zebrafish, RNA sequencing * Correspondence: 1 Department of Biomolecular Medicine & Center for Medical Genetics, Ghent University, 9000 Ghent, Belgium 2 Cancer Research Institute Ghent (CRIG), 9000 Ghent, Belgium Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Loontiens et al. BMC Genomics (2019) 20:228 Background Over the recent years, it has become clear that transcriptional control relies on the orchestrated activity of transcription factor complexes, DNA methylation, chromatin modification dynamics as well as higher-order DNA looping [1]. Genetic lesions and epigenetic alterations have a major impact on rewiring of transcriptional networks during cancer development and importantly mining these perturbed transcriptomes allows to uncover novel therapeutic targets [2–4]. Technological advances in the field of massively parallel sequencing have greatly facilitated in-depth assessment of gene expression programs, with RNA sequencing presently serving as the gold standard for detailed and unbiased molecular characterization of both in vitro and in vivo model systems. In recent years, zebrafish (Danio rerio) has become increasingly important as a model organism to study vertebrate development and cancer [5], as many cellular processes and developmental programs are evolutionary conserved. Furthermore, embryonic development in zebrafish is fast, with completion of embryogenesis within 5 days and adulthood reached in 3 months. The short generation time and large progeny combined with the ease to (non-invasively) study transparent embryos present obvious advantages compared to murine in vivo models [6, 7]. While zebrafish was initially primarily used for developmental studies, it is now also emerging as a relevant model to study human diseases [8–12]. The development of fluorescent reporter zebrafish lines, where a fluorescent marker is driven by a cell type-specific promoter, makes it feasible to perform fluorescence activated cell sorting (FACS), thereby circumventing the need of zebrafish specific antibodies for staining of specific cell populations. In this manner, straightforward enrichment of the cells of interest is feasible, enabling the definition of cell-specific transcriptomes that could otherwise be masked when ‘whole embryo’ derived RNA is analysed [13–16]. Despite the above-mentioned advantages, some considerations have to be made when using these zebrafish reporter lines for RNA-sequencing purposes. First, cells undergoing the process of sorting encounter stress and show reduced viability [17]. In dying cells, RNA decay is triggered and therefore inappropriate handling of the sample adds onto this RNA degradation process. The resulting transcriptome may therefore not be representative for the in vivo gene expression levels. In addition, RNA degradation does not occur at the same pace for every transcript and is defined by different aspects of the RNA transcript sequence (such as GC content, and length of the coding DNA sequence) [18–21]. This makes it very important to optimize a workflow that minimizes RNA degradation due to FACS induced cellular stress and cell death to obtain high quality RNA for expression analysis. Also, purified RNA is frequently contaminated with genomic DNA, Page 2 of 16 possibly impacting on expression analysis results. Finally, sorting a large number of cells (> 1 million) is not always feasible when working with fluorescent reporter embryos. Depending on the promotor of choice, the fluorescent marker may only be expressed in a small number of cells and thereby result in a low RNA yield. Given the importance obtaining sufficient amounts of high quality RNA for expression studies, we optimized RNA extraction from FACS sorted cells from zebrafish embryos and did an in-depth quality assessment of the extracted RNA, the latter often omitted in performed experiments. We used the Tg(fli1a:GFP) zebrafish line to optimize our workflow. This zebrafish line ex (...truncated)