(*contributed equally to this work) Published: Vol 10, Iss 21, Nov 5, 2020 DOI: 10.21769/BioProtoc.3809 Views: 3059
Reviewed by: Lijuan DuBenjamin HousdenAnonymous reviewer(s)
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Abstract
Cell-type specific transcriptional programs underlie the development and maintenance of organs. Not only distinct cell types within a tissue, even cells with supposedly identical cell fates show a high degree of transcriptional heterogeneity. Inevitable, low cell numbers are a major hurdle to study transcriptomes of pure cell populations. Here we describe DigiTAG, a high-throughput method that combines transposase fragmentation and molecular barcoding to retrieve high quality transcriptome data of rare cell types in Drosophila melanogaster. The protocol showcases how DigiTAG can be used to analyse the transcriptome of rare neural stem cells (type II neuroblasts) of Drosophila larval brains, but can also be utilized for other cell types or model systems.
Keywords: RNA SequencingBackground
Transitions between different cell types during development and tissue homeostasis are orchestrated by a plethora of transcription factors and their induced transcriptional changes. In the last decade, RNA sequencing (RNA-seq) has become the classical approach to measure transcriptional dynamics across the genome (Stark et al., 2019). Bulk RNA-seq on tissues does not allow to investigate transcriptional networks of different cell populations, specifically those of rare cell types. Thus, RNA-seq protocols that deliver high quality transcriptomes of low input samples are required.
In Drosophila, limited material often constitutes a hurdle to analyse particular tissues or cell types. This is well illustrated by Drosophila neural stem cells, called neuroblasts (Homem and Knoblich, 2012). Several distinct subpopulations of neuroblasts exist. For example, in the larval Drosophila brain only sixteen type II neuroblasts produce neurons innervating brain regions required for locomotion and sensory processing (Walsh and Doe, 2017). Defects in type II neuroblasts do not only result in aberrant neurogenesis but can also induce tumor growth (Knoblich, 2010).
To overcome low cell numbers, mutants displaying tumor-like overgrowth of particular neuroblast subpopulations have been used for bulk RNA-seq to enrich for cells-of-interest (Carney et al., 2012). Although such an approach has given insights into the heterogeneity of neural stem cells, it eliminates the possibility to distinguish between tumor- and cell type-specific changes. Cell isolation by flow cytometry (Berger et al., 2012; Harzer et al., 2013) or by robotic single-cell picking (Yang et al., 2016) have greatly advanced the purity of the isolated cell populations, but manual dissection or the navigation of special equipment still limit cell numbers. Recently, single-cell RNA-seq has been used to map the Drosophila brain (Brunet Avalos et al., 2019), however such high resolution and complex datasets might not be required to answer many scientific questions and is suboptimal due to costs, non-standard analysis, higher technical noise and loss of lowly expressed genes. Therefore, we present here a RNA-seq protocol for Drosophila neuroblasts that can be performed in any standard molecular biology laboratory using commercially available reagents (Figure 1). First, RNA is isolated and polyA-containing RNA molecules are converted into double-stranded cDNA (Figure 1A). Then the simultaneous cDNA fragmentation and ligation of sequencing adapters to each cDNA molecule by the use of transposases (Figure 1B), is time-efficient and highly reproducible. Primers tagged with unique barcodes are subsequently used to amplify the fragmented cDNA molecules and allow the identification of PCR biases, which can be removed during analysis (Figure 3). The combination of transposon-mediated library preparation with molecular barcoding to quantify the original library molecules rather than their amplicons (Figure 1) ensures high quality of the transcriptome data. Recently, DigiTAG has been successfully used to compare tumor cells to their rare cells-of-origin (Landskron et al., 2018), identify growth regulators of differently sized cells (Wissel et al., 2018) and decipher the temporal patterning of transient-amplifying progenitors (Abdusselamoglu et al., 2019).
Figure 1. Overview of the DigiTAG workflow. A. RNA is isolated from the purified cells-of-interest and converted into double-stranded cDNA. To generate the final sequencing library, cDNA molecules are fragmented and amplified (for more detail see B). B. Generation of sequencing libraries from cDNA. By using transposases, cDNA is simultaneously fragmented and adapters (dark grey and brown) are ligated. In the following amplification step these adapters serve as binding sites for two types of primers. Forward primers include the Illumina flow cell attachment sequence P5 (red) together with a sample-specific index sequence (green, allowing to multiplex several RNA-seq samples if desired), whereas reverse primers contain the Illumina flow cell attachment sequence P7 (purple) with a molecule-specific index sequence (rainbow-colored, allowing to barcode each amplified molecule uniquely).
Materials and Reagents
Hardshell 96-Well PCR Plate, low profile, thin wall, skirted, white/clear (Bio-Rad, catalog number: HSP9601 )
Microseal ‘B’ PCR Plate Sealing Film, adhesive, optical (Bio-Rad, catalog number: MSB1001 )
Refrigerated centrifuge
DNA LoBind tubes 1.5 ml (Eppendorf, catalog number: 0030108051)
Filter tips for micropipettes
Drosophila
TrizolTM LS (ThermoFisher Scientific, catalog number: 10296028 ) (Storage: 4 °C until expiration date)
Nuclease-free Water (ThermoFisher Scientific, catalog number: AM9932 ) (Storage: Room temperature until expiration date)
InvitrogenTM GlycoBlueTM Coprecipitant (ThermoFisher Scientific, catalog number: AM9515 ) (Storage: -20 °C until expiration date)
Chloroform (Sigma-Aldrich, catalog number: C2432 , handle in fume hood!)
70% and 75% ethanol, prepared from absolute Ethanol (Sigma-Aldrich, catalog number: 32205 , store at room temperature in fireproof cabinet), diluted with Nuclease-free water.
Isopropanol (Sigma-Aldrich, catalog number: 278475 , store at room temperature in fireproof cabinet)
Oligo(dT)20 (50 μM) (ThermoFisher Scientific, catalog number: 18418020 ) (Storage: -20 °C until expiration date)
dNTPs (10 mM each) (ThermoFisher Scientific, catalog number: R0193 ) (Storage: -20 °C until expiration date)
5x First Strand Buffer, DTT (100 mM) and SuperScript III (200 U/μl) are all part of the kit: SuperScriptTM III Reverse Transcriptase (ThermoFisher Scientific, catalog number: 18080093 ) (Storage: -20 °C until expiration date)
MgCl2 (25 mM) (ThermoFisher Scientific, catalog number: R0971 ) (Storage: -20 °C until expiration date)
RNaseOUT (40 U/μl) (ThermoFisher Scientific, catalog number: 10777019 ) (Storage: -20 °C until expiration date)
5x Second Strand Buffer (ThermoFisher Scientific, catalog number: 10812014 ) (Storage: -20 °C until expiration date)
RNase H (5 U/μl) (ThermoFisher Scientific, catalog number: EN0202 ) (Storage: -20 °C until expiration date)
DNA Polymerase I (10 U/μl) (ThermoFisher Scientific, catalog number: 18010017 ) (Storage: -20 °C until expiration date)
AMPure XP Beads (Beckman Coulter, catalog number: A63880 ) (Storage: 4 °C until expiration date)
EB Buffer (Qiagen, catalog number: 19086 ) (Storage: Room temperature until expiration date)
2x Phusion HF master mix (ThermoFisher Scientific, catalog number: F531S ) (Storage: -20 °C until expiration date)
TDE Tagment DNA buffer and TDE1 Tagment DNA enzyme are all part of the kit: Illumina Tagment DNA TDE1 Enzyme and Buffer Kits (Illumina, catalog number: 20034197 ) (Storage: -20 °C until expiration date)
20x Eva Green (Biotum, catalog number: 31000 ) (Storage: -20 °C until expiration date)
Nextera PCR, modified Index one and Index two primers are all part of the kit: Nextera DNA Library Preparation Kit (24 samples) (Illumina, catalog number: FC-121-1030 ) (Storage: -20 °C until expiration date)
Real-Time Standard 1-4 are all part of the kit: KAPA Real-time Library Amplification kit (KAPA Biosystems, catalog number: KK2709 ) (Storage: -20 °C until expiration date)
Equipment
Touch Real-Time PCR Detection System (Bio-Rad, model: CFX96 )
Thermomixer Compact with 1.5 ml block (Eppendorf, model: Thermomixer Compact )
Magnet rack (Fisher Scientific, InvitrogenTM DYNALTM DynaMagTM DynabeadsTM DynaMag-2 Magnet, catalog number: 10723874 )
0.2-2 μl micropipette
10-100 μl micropipette
20-200 μl micropipette
100-1,000 μl micropipette
Software
CFX MaestroTM Software (Bio-Rad)
Procedure
Isolation of cells-of-interest
The cell type-of-interest can be isolated through various different methods. For Drosophila neuroblasts, isolation by flow cytometry is recommended. Briefly, Drosophila brain cells as for example type II neuroblasts cells are labeled using fluorophore-expressing driver lines (e.g., worniu-GAL4, ase-GAL80, UAS-CD8::GFP for type II neuroblasts). Brains are manually dissected and dissociated using enzymatic treatment followed by mechanical disruption. Single-cell suspensions are then subjected to flow cytometry. Neuroblasts identified as a separate cell population, which includes the largest GFP positive cells, are isolated and either stored at -80 °C in TrizolTM or immediately used for RNA isolation. For a step-by-step protocol see Harzer et al. (2013).
RNA isolation
Reverse Transcription
Second Strand Synthesis
DNA Purification
Tagmentation
PCR amplification
Data analysis
Summary of the analysis
Most critical analysis steps are the following (see also Figure 3): In a first step, sequencing reads are aligned to the genome. Ribosomal RNA reads are subtracted from all aligned reads. Next, reads that arose due to biased amplification (in the step shown in Figure 1B) are identified using the molecule-specific barcode. At each position in the genome the different barcodes are counted. Reads sharing the same barcode are marked as “duplicated reads”. Only the read with the highest quality (this can be assessed with the PHRED score) is kept while all others are removed. The aligned and deduplicated reads are then counted and polyA-containing transcripts are subjected to a differential expression analysis (for example DESeq, Anders and Huber, 2010).
Figure 3. Barcoding strategy for data analysis. The sequencing library (see also Figure 1) contains DNA molecules with sequences allowing the attachment to the flow cell (P5/P7) and indices. The sample-specific index (green) can be used to pool several sequence libraries for a single sequencing run (multiplexing). The molecule-specific barcode is unique for each fragmented, double-stranded DNA molecule. Sequencing reads are mapped to the reference genome. The molecule-specific barcodes identify PCR amplicons derived from the same DNA molecule and thus allow the correction of such PCR bias by filtering duplicates.
Step by step procedure of the RNA-seq data analysis
Further documentation of the analysis can be found in the method section of Landskron et al. (2018) and all data are available on this link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE87085.
Notes
Always use 1.5 ml DNA LoBind tube along the protocol to limit DNA loss.
Always use Nuclease-free Water to prepare ethanol solutions.
Acknowledgments
We would like to particularly thank Jonas Steinman and Heike Harzer for pioneering DigiTAG. This work was funded by the Austrian Academy of Sciences, Austrian science Fund and the European Commission. F.B. was supported by the European Molecular Biology Organization. This protocol is derived from Landskron et al. (2018).
Competing interests
Authors declare no competing interests.
References
Article Information
Copyright
Landskron et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
How to cite
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
Category
Developmental Biology > Cell growth and fate > Neuron
Developmental Biology > Cell signaling > Fate determination
Molecular Biology > DNA > DNA sequencing
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