神经元突起RNA含量的Axon-seq 深入分析

Jik Nijssen; Julio Aguila; Eva Hedlund

doi:10.21769/BioProtoc.3312

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Peer-reviewed

Axon-seq for in Depth Analysis of the RNA Content of Neuronal Processes

神经元突起RNA含量的Axon-seq 深入分析

Jik Nijssen

JA Julio Aguila

Eva Hedlund email

发布: 2019年07月20日第9卷第14期 DOI: 10.21769/BioProtoc.3312 浏览次数: 11537

评审: Gal HaimovichShyam SolankiAnonymous reviewer(s)

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Abstract

Neuronal processes have an RNA composition that is distinct from the cell body. Therefore, to fully understand neuronal biology in health and disease we need to study both somas, dendrites and axons. Here we describe a detailed protocol of a newly refined method, Axon-seq, for RNA sequencing of axons (and dendrites) grown in isolation using single microfluidic devices. We also detail how to generate motor neurons from mouse and human pluripotent stem cells for sequencing, but Axon-seq is applicable to any neuronal cell. In Axon-seq, the axons are recruited through a growth factor gradient, lysed and directly processed to cDNA without RNA isolation. A careful bioinformatic step ensures that any soma-contaminated samples are easily identified and removed.

Keywords: RNA sequencing (RNA测序)

Motor neuron (运动神经元)

Axons (轴突)

Stem cells (干细胞)

Microfluidic devices (微流控装置)

Bioinformatics (生物信息学)

Background

Neurons are highly polarized cells. Their processes, both dendrites and axons, need to be able to respond to changes in the microenvironment in a manner independent of the soma (Holt and Schuman, 2013). To fully understand neuron biology, it is therefore important to be able to study neurites in isolation, in addition to conducting analysis of the cell bodies. This appears particularly important for neurons where the axon and dendrites constitute the majority of the cellular volume, as is the case for spinal motor neurons where we estimate that they comprise approximately 99% of the cellular volume. To isolate neurites, there are excellent tools such as Campenot Chambers (Boyden, 1962; Campenot, 1977) or microfluidic chambers (Taylor et al., 2005).

However, while neurites are separated out in such devices, it is important to bear in mind that cross-contamination between compartments can still occur. Thus, RNA sequencing of isolated axonal compartments (Minis et al., 2014; Saal et al., 2014; Briese et al., 2016; Rotem et al., 2017) can lead to incorrect results/conclusions when the purity and exclusion of somas is insufficiently examined. To ensure detailed and accurate investigation of motor axonal mRNA composition and its modulation in ALS we developed Axon-seq (Nijssen et al., 2018). This is an application of our LCM-seq method for single-cell spatial RNA-sequencing (Nichterwitz et al., 2016).

In Axon-seq, we use microfluidic devices to separate axons from stem cell-derived motor neurons (mouse and human) from their somas. As motor neurons have axons that traverse far longer distances than their dendrites, we are able to analyze axons alone. This may not be possible for all neuronal subtypes, but the method still allows for analysis of neurites as an entity.

In contrast to previous methods, Axon-seq does not require an RNA isolation step, and it allows for high sensitivity and cost-efficient sequencing from a single microfluidic device.

Notably, Axon-seq effectively eliminates all samples with any somatic cross-contamination, as it uses a highly stringent and sensitive bioinformatic quality control step that identifies axonal samples containing trace levels of mRNA from undesired cell somas. Here we provide a detailed protocol for Axon-seq which can be applied to any cell containing longer processes.

Materials and Reagents

Materials
1. Sterile 6 and 10 cm bacterial dishes (non-adhesive plastic) (Corning, catalog numbers: 351007 and 351029)
2. Pipette tips (0.5-10 μl, 10-100 μl, 100-1,000 μl)
3. Sterile 15 and 50 ml Falcon tubes
4. Parafilm
5. 70 μm cell-strainer filter (VWR, catalog number: 732-2758P)
6. 24 x 32 mm cover glasses (VWR, Menzel Gläser, catalog number: 630-2873)
7. Soft pencil-type brush (Any soft brush type that does not have too strong hairs that could pierce/damage the PDMS-surface of which the microfluidic devices are made. Brushes, from a typical hardware/convenience store, with a diameter of 2-5 mm work best, laboratory grade is not required. Nylon/synthetic hairs are not an issue.)
8. Waterproof marker with fine (1 mm) or very fine (0.3 mm) tip
Biological material
1. Motor neurons from mouse embryonic stem cells
2. Motor neurons from human pluripotent stem cells
Reagents
1. 70% ethanol
2. Triton X-100 (Sigma-Aldrich, catalog number: X100)
3. PBS (Thermo Fisher, catalog number: 14190136)
4. Poly-L-ornithine, 0.01% solution (Sigma-Aldrich, catalog number: A-004-C)
5. Fibronectin, 1 mg/ml solution (Sigma-Aldrich, catalog number: F1056)
6. Laminin, 1 mg/ml solution (Sigma-Aldrich, catalog number: L2020)
7. TrypLe Express (Thermo Fisher, catalog number: 12604021)
8. Neurobasal (Thermo Fisher, catalog number: 21103049)
9. DMEM/F12 with GlutaMAX (Thermo Fisher, catalog number: 31331028)
10. B27 supplement, custom (Thermo Fisher, catalog number: 0080085SA)
11. Penicillin/Streptomycin (Thermo Fisher, catalog number: 15140122)
12. GlutaMAX supplement 100x (Thermo Fisher, catalog number: 35050061)
13. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M6250)
14. Ascorbic acid, 100 mg powder (Sigma-Aldrich, catalog number: A4403), prepare as 200 mM stock solution by dissolving the powder in 2.84 ml of nuclease-free dH₂O
15. Y-27632, 10 mg powder (Tocris, catalog number: 1254), prepare as 10 mM stock solution by dissolving the powder in 3.12 ml nuclease-free dH₂O
16. LDN-193189 dihydrochloride, 10 mg powder (Tocris, catalog number: 6053), prepare as 10 mM stock solution by dissolving the powder in 2.09 ml DMSO, then prepare 1 mM working stocks (1:10 diluted) in DMSO
17. SB-431542, 10 mg powder (Tocris, catalog number: 1614), prepare as 10 mM stock solution by dissolving the powder in 2.60 ml DMSO
18. CHIR-99021, 10 mg powder (Tocris, catalog number: 4423), prepare as 3 mM stock solution by dissolving the powder in 7.16 ml DMSO
19. DAPT, 10 mg powder (Tocris, catalog number: 2634), prepare as 10 mM stock solution by dissolving the powder in 2.31 ml DMSO
20. SAG, 1 mg powder (Tocris, catalog number: 4366), prepare as 500 μM stock solution by dissolving the powder in 4.08 ml DMSO
21. All-trans retinoic acid, 50 mg powder (Sigma-Aldrich, catalog number: R2625), prepare as 100 mM stock solution by dissolving the powder in 1.66 ml DMSO, then prepare 1 mM working stocks (1:100 diluted) in DMSO
22. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D2650)
23. Bovine serum albumin (BSA), 7.5% solution in dPBS (Sigma-Aldrich, catalog number: A8412)
24. Glial-derived neurotrophic factor (GDNF), 10 μg powder (Peprotech, catalog number: 450-10), prepare as 10 μg/ml stock solution by dissolving the powder in 1 ml dPBS with 0.1% BSA
25. Brain-derived neurotrophic factor (BDNF), 10 μg powder (Peprotech, catalog number: 450-02), prepare as 10 μg/ml stock solution by dissolving the powder in 1 ml dPBS with 0.1% BSA
26. RNase-inhibitor (Takara, catalog number: 2313A)
27. DTT (part of SuperScript II Reverse Transcriptase kit, Thermo Fisher, catalog number: 18064071)
28. Nuclease-free dH₂O (Thermo Fisher, catalog number: 10977-035)
29. 37% formaldehyde solution (Merck Millipore, catalog number: 818708)
30. Mouse MN medium (see Recipes)
31. N2 medium (see Recipes)
32. B27 medium (see Recipes)
33. Human EB medium (see Recipes)
34. Lysis/harvesting solution (see Recipes)

Equipment

Glass beaker (500 ml)
Sterile forceps (Dumont, catalog number: 11251-20, or other forceps of similar standard size)
Pipettes (0.5-10 μl, 10-100 μl, 100-1,000 μl)
Magnetic stirrer
Sterile laminar flow hood
Humidified incubator (37 °C, 5% CO₂)
Suction aspirator
Standard Neuron Device (Xona Microfluidics^TM, SND75/150/450/900)
Low-speed orbital shaker (Elmi-tech, DOS-20S)
Inverted bright field microscope (> 20x magnification is required to reliably see axons exiting the microgrooves. Examples are Laxco LMI-3000 or Carl Zeiss PrimoVert. Any similar setup is fine.)

Software

R (https://www.r-project.org)
RStudio open source edition, Boston, MA, USA (https://www.rstudio.com)

Procedure

English

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Nijssen, J., Aguila, J. and Hedlund, E. (2019). Axon-seq for in Depth Analysis of the RNA Content of Neuronal Processes. Bio-protocol 9(14): e3312. DOI: 10.21769/BioProtoc.3312.