本文章节 AbstractBackgroundMaterials and ReagentsEquipmentSoftwareProcedureData analysisRecipesAcknowledgmentsCompeting interestsEthicsReferences 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 NijssenAffiliation: Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 查看作者页面Julio AguilaAffiliation: Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 查看作者页面Eva Hedlund eva.hedlund@ki.seAffiliation: Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden 查看作者页面 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.

Sterile 6 and 10 cm bacterial dishes (non-adhesive plastic) (Corning, catalog numbers: 351007 and 351029) 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.) 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 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 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 SB-431542, 10 mg powder (Tocris, catalog number: 1614), prepare as 10 mM stock solution by dissolving the powder in 2.60 ml DMSO CHIR-99021, 10 mg powder (Tocris, catalog number: 4423), prepare as 3 mM stock solution by dissolving the powder in 7.16 ml DMSO DAPT, 10 mg powder (Tocris, catalog number: 2634), prepare as 10 mM stock solution by dissolving the powder in 2.31 ml DMSO SAG, 1 mg powder (Tocris, catalog number: 4366), prepare as 500 μM stock solution by dissolving the powder in 4.08 ml DMSO 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 Bovine serum albumin (BSA), 7.5% solution in dPBS (Sigma-Aldrich, catalog number: A8412) 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 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 DTT (part of SuperScript II Reverse Transcriptase kit, Thermo Fisher, catalog number: 18064071) Sterile forceps (Dumont, catalog number: 11251-20, or other forceps of similar standard size) 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.) RStudio open source edition, Boston, MA, USA (https://www.rstudio.com) Storage and washing of microfluidic devicesCommercial microfluidic devices from Xona MicrofluidicsTM (Standard Neuron Devices) with any groove length can be stored in dH2O and used multiple times. Note: The groove length of the devices could affect the proportion of axons versus dendrites that can cross over to the opposite chamber, as could the length of time of culture in the devices. For mouse motor neurons, we used devices with 150 μm length grooves and saw a clear majority of axons being recruited during the 7 days of culture. For human motor axons, we used devices with 450 μm grooves. Place devices in a glass beaker with 500 ml of 1% Triton X-100 in dH2O for 2 h and use a magnetic stirrer to keep devices agitated. Use a soft brush to gently clean the microgroove surfaces and the chambers of the devices. Transfer devices into dH2O afterward. Wash 3 x 10 min in fresh dH2O each time to remove any residues of Triton X-100. Then, sterilize devices in 70% ethanol for a minimum of 10 min.Note: The washing procedures can be done at any time and devices can be stored in 70% ethanol to be ready when needed. Start the device attachment (below) the day that the coating procedure is to be initiated. Remove microfluidic devices from 70% ethanol using sterile forceps and allow the devices to air-dry in a sterile laminar flow hood for 20 min. Use a suction aspirator to remove any residual ethanol.Note: It is very important that devices are entirely dry at this stage, as any remaining liquid will negatively affect device attachment and can cause leakage in later stages. Place sterile glass cover glasses (32 x 24 mm, thickness #1 or #1.5) on top of the water drops to ensure that the cover glasses do not move around within the dish (Figure 1B). Use forceps to pick up one device at a time and gently press it, feature side down, onto the glass coverslip (Figure 1C). Apply gentle force, using the back of the forceps, to the center of the device (above the grooves), and all four corners. A microscope can be used to assess if all the grooves are in the same focal plane and thus attached to the coverslip. Devices attached in this way are stable for at least four weeks (and likely longer). Label the glass coverslips with a waterproof marker in one corner to define the future left-right orientation of the devices. The device forms the ‘ceiling’ for the cells and creates grooves for the axons to grow through. Cells will attach to the glass surface of the coverslip. The final setup of devices (Step B4) is a suggestion. Devices can also be placed directly in tissue culture-grade plastic dishes (i.e., directly onto the plastic, without cover glasses). For imaging purposes, however, placing them on glass coverslips is beneficial. In addition to the PDMS devices described here, Xona MicrofluidicsTM also offers pre-bonded devices for cell culture use (XonaChips). While these cost the same as regular devices, they are not optimal for reusing, as can be done with the regular devices (SND150). In the case of pre-bonded devices, the attachment step (Procedure B) can be skipped.Figure 1. Setup of 4 microfluidic devices in a 10 cm dish. A. First, place 4 drops of 4 μl dH2O spaced apart evenly in the 10 cm dish. Then place glass cover glasses onto the drops to create a water seal between cover glasses and the plastic dish. B. When cover glasses are attached, place one microfluidic device with feature-side down on each cover glass. Press gently on the top of the device to ensure attachment. C. Final setup of four devices in one 10 cm dish. Other arrangements of devices (in terms of dish size) can be made if desired. The primary surface coating consists of 0.001% poly-L-ornithine in dH2O. Prepare 0.5 ml solution per device. Add 75 μl solution into the two bottom wells of each device. Typically, the liquid will quickly fill the chambers through capillary forces. If this does not happen, use the aspirator with a mounted 1 ml pipette tip to gently suck the liquid through from the top. Do not aspirate all the liquid. Once the liquid has flown through, add 150 μl to the top left and 200 μl to the top right compartment. Adding different volumes between the left and right chamber ensures that the coating is forced into the microgrooves over time. Leave the coating on overnight at room temperature and shield it from light (e.g., a drawer works well). Wash the devices twice using 1x PBS. To wash, aspirate the coating from the wells, then add PBS into the wells, 75 μl in the bottom, then 150 μl in the top. The second coating consists of a solution of 2 μg/ml fibronectin and 5 μg/ml laminin in PBS. Add this last coating in a similar fashion to the first–see Steps C2-C4. Specification of motor neurons from mouse embryonic stem cellsTypically, a good starting point for differentiation is a 10 cm dish of stem cells at ~70-80% confluency. Cells are always maintained in a humidified incubator at 37 °C and 5% CO2.Day 1: Prewarm TrypLE Express to be used for dissociation at 37 °C (i.e., 2 ml for a 6 cm dish, 4 ml for 10 cm dish) in a tube. In addition, pre-warm a tube with 8 ml DMEM/F12-GlutaMAX (or any other plain MEM-based media). When cells have rounded up and are coming off the culture dish, use a pipette to dissociate them fully in the TrypLE Express, then transfer everything into the tube with pre-warmed DMEM. Remove supernatant and resuspend cells in 1 ml of mouse MN media (Recipe 1) and count. Place cells in bacterial (non-adherent) dishes at a density of 0.5-1.5 million cells per ml (the number of cells is line dependent and needs to be tested out), and place dishes on a low-speed orbital shaker at 30 rpm overnight in the incubator. Remove dishes from the shaker and change to fresh mouse MN media. To change media on EBs, pipette the entire volume of media (including EBs) into a tube. Allow the EBs to sink for ~2 min and remove the supernatant. Resuspend EBs in 1 ml fresh media and place into a new culture dish. Change to fresh mouse MN media, supplemented with 100 nM all-trans-retinoic acid (RA) and 500 nM smoothened agonist (SAG). Repeat for 4 successive days. See Table 1.Table 1. Factors for motor neuron differentiation from mouse pluripotent stem cells After 4 consecutive days of patterning, cells are ready to be dissociated and seeded into microfluidic devices (see below). Specification of motor neurons from human pluripotent stem cellsTypically, a good starting point for differentiation is a 10 cm dish of stem cells at ~70-80% confluency. Cells are always maintained in a humidified incubator at 37 °C and 5% CO2.Day 1:The procedures on the first day (EB formation) are the same as described above for mouse ES cells. However, in the end, resuspend the cells in human EB media at a density of 0.5 M-0.75 M cells per ml (this density may require a degree of optimization between cell lines). Supplement the Day 1 human EB media with the required factors for Day 1 (for overview, see Table 2):5 μM Y-27632 (ROCK-inhibitor)200 nM LDN-19318940 μM SB-4315423 μM CHIR-99021200 μM ascorbic acidTable 2. Factors for motor neuron differentiation from human pluripotent stem cellsDays 2-10:Perform daily media changes at approximate 24 h intervals with the corresponding factors for each day (Table 2).Note: During the RA-SAG stage of the protocol (days 3-10), it is not absolutely required that media changes follow 24 h intervals, and if necessary (but not recommended) one day of media changes can be omitted. However, it is critical that the dual-SMAD inhibition (using SB-431542 and LDN-193189) during the first two days is 48 h and not shorter.Day 10:Cells are ready to be dissociated and seeded into microfluidic devices (see below). Transfer EBs into a sterile 15 ml Falcon tube. Allow the EBs to sink to the bottom for 1-5 min and aspirate the remaining media. Add 1 ml pre-warmed (37 °C) TrypLE Express to the Falcon tube with EBs. The dissociation takes 10-20 min, depending on the size of the EBs. Gently agitate the EBs every 5 min by tapping the tube or gently pipetting up and down using a 1 ml pipette.Note: If you resuspend EBs by pipette for the first time after 5 min in TrypLE Express, do not pipette them up into the pipette tip as they tend to stick on the inside. Instead, just pipette the liquid and ensure the EBs consequently are agitated slightly in the suspension. Once the EBs are beginning to break apart, pipette them up and down rather vigorously approximately 10 times to finalize the dissociation. Add 9 ml of pre-warmed DMEM to dilute the 1 ml of TrypLE Express. Filter the resulting suspension through a 70 μm cell-strainer filter to remove undissociated clumps/debris, then spin at 200 x g for 4 min, at room temperature. Resuspend the pellet in 1 ml of DMEM/F12-GlutaMAX (or any other plain MEM-based media). Count the cells. Adjust the density to a final concentration of 2.5 x 104 cells per μl (this will ensure that a total of 1 x 105 cells are seeded per microfluidic device later on). To do this, transfer 1 ml of the cell suspension into an Eppendorf tube and spin once more at 200 x g for 4 min. Resuspend cells in the required amount of media (i.e., 1 μl for every 2.5 x 104 cells). At this stage, the cells should be resuspended in the final culturing media, for human cells this is B27 media with the Day 10 factors (see Table 2), for mouse cells this is mouse MN media with Day 7 factors (see Table 1). Note: The exact composition of the final media can vary based on the type of neurons in culture. However, the addition of ascorbic acid and trophic factors (and ROCK-inhibitor for human cells) is highly recommended to improve survival upon plating in the confined environment of the microfluidic device. Prepare sufficient final media to fill all devices (0.5 ml per device). In the end, the device will hold media volumes as shown in Figure 2. Take the required amount of media (see Figure 2) for the axonal compartment (top and bottom well) and transfer to a new tube. Add trophic factors to increase the concentration to 50 ng/ml. The higher concentration of trophic factors on the \"axonal” side will improve axonal recruitment across the microgrooves.Figure 2. Media volumes and trophic factor concentrations in the microfluidic device at seeding. Note that somatic concentrations of the neurotrophic factors differ between human and mouse MNs. After axons have crossed the microgrooves into the other compartment, trophic factor concentrations can be equalized to 10 ng/ml on both sides, both for mouse and human. Start removing coating from the devices by aspiration (from one 10 cm dish with four devices at a time). Add 4 μl of the axonal media (containing 50 ng/ml BDNF/GDNF) into the chamber on the axonal side. Pipette close to the chamber entrance on the top-left (in the orientation of Figure 2, see Figure 3A for photograph) and allow capillary forces to pull the liquid into the chamber. Add the cells in the same manner to the other compartment. Once cells are seeded in the chamber, place devices in the incubator and allow the cells to attach for approximately 15-20 min. Start checking devices from 10 min after seeding the cells and onward to avoid excessive evaporation of the media. Once cells appear attached, add the final media amounts to the wells as indicated in Figure 2. Check if the cells remain attached and then place devices in the incubator overnight.Notes: Occasionally cells do not attach well even after 15 min, maybe in part due to the flow between the compartments. If this occurs, we recommend adding only 40 μl of media to all wells (keeping the right media types for each side), which will avoid excessive flow between compartments. This way, there is minimal flow and evaporation will not be an issue. Then keep the cells like this for an additional hour and finally add media to the recommended amounts in Figure 2. For human cells, the dissociation takes place on Day 10 of the protocol. In addition to everything described here, maintain the corresponding factors in the media for each day as described in Table 2. The trophic factor (BDNF/GDNF) concentrations described there are for the somatic compartment, the axonal compartments receive the trophic factor gradient for the first few days (see below).Figure 3. Seeding cells into microfluidic chambers and representative micrographs. A. Example photographs of the angle at which media/cells can be added into the chambers of the microfluidic device. Dashed lines show the location of the chambers, and chamber entrances. B. Micrographs of a good culture in the axonal compartment and the somatic compartment, where clusters of neuron somas are visible. After seeding neurons into microfluidic devices, change media daily to ensure that the trophic factor gradient stays intact. A bright field microscope can be used to keep track of the axonal growth.Note: When changing media, do NOT aspirate media out of the chambers as this will forcibly remove all cells/axons, only change media in the wells. Once axons can be seen crossing several microgrooves into the axonal compartment (i.e. 10 grooves with axons), the trophic factor gradient can be removed, and both compartments instead receive 10 ng/ml of GDNF and BDNF. At this point, media can be exchanged every other day. See Figure 3B for a successful micrograph of a microfluidic device with growing axons. Prepare the lysis/harvesting solution. Prepare ~70 μl solution per microfluidic device, or less if only one compartment is harvested (final recommended lysis volumes are 50 μl for the somatic compartment and 10 μl for the axonal compartment). The lower volume for axons ensures that a higher concentration of RNA is present in the final solution, which aids in downstream applications. The lysis solution (see Recipes) is made up as a 2% Triton X-100 solution in nuclease-free dH2O. First, cool this solution on ice, then supplement with:0.5 μl RNase-inhibitor per 100 μl (final concentration of 0.2 U/μl)1 mM dithiothreitol (DTT), required for RNase-inhibitor enzymatic activityNote: Always keep lysis solution on ice during preparation and use. Prepare nuclease-free Eppendorf tubes. Label them according to the samples that are being collected, and pre-cool them on ice. Have dry ice ready to snap-freeze harvested samples immediately following the collection. Optionally, before starting the harvesting, take bright field images along the grooves. If somatic contamination is detected in the downstream bioinformatics analysis of axon samples, these images can be scrutinized for evidence of any cross-contaminating somas. Harvest one device at a time. Begin by washing both compartments once with pre-warmed (37 °C) PBS. Use at least 100 μl PBS. Remove PBS from the axonal wells and add 10 μl lysis solution to the top of the chamber, as if seeding cells (the vastly higher volume of PBS on the somatic side prohibits any lysis solution from flowing through the grooves and accidentally lyse somas). Wait for 10 s, then pipette up and down ~5 x in the bottom well, and extract as much solution as possible from the bottom of the chamber (can be 10 μl). Transfer solution into a prepared Eppendorf tube and snap-freeze on dry ice. Repeat Steps I7 and I8 above for the somatic side, but instead use 50 μl lysis solution. When extracting the solution, try to extract as much as possible ( 50 μl). Snap-freeze on dry ice. After collection and snap-freezing, samples for RNA sequencing can be stored at -80 °C until further used.Note: For detailed instructions, protocols and reagents for the preparation of RNA sequencing libraries without RNA-extraction, see Nichterwitz et al., 2018. To generate axonal and somatodendritic sequencing libraries, standard laboratory practices related to handling of RNA must be considered. Namely, keep the samples cold (4 °C) and surfaces clean. Work with RNAse/DNAse-free disposables and reagents certified for molecular biology grade. Consider establishing a dedicated working station (bench) and a thermocycler exclusive for library preparation purposes. Due to the low amount of material (RNA) per device, comparable to that of single cells, we employed the Smart-seq2 protocol, an established methodology for single-cell transcriptomics. Briefly, in the first step, samples are quickly thawed from -80 °C and 10 μl of axonal lysate (5 μl of somatodendritic lysate) are subjected to reverse transcription using an oligo-dT primer and a template switch LNA-oligo. In the second step, an enrichment PCR with 18 cycles is introduced to amplify the amount of cDNA and samples purified using magnetic beads. At this point, the quality and quantity of the cDNA libraries can be measured by bioanalyzer using a dsDNA high sensitivity chip (for representative examples, see Figure 4). Finally, cDNA libraries are tagmented (Nextera XT Kit, Illumina) and barcoded with Illumina indexes for RNA sequencing.Figure 4. Typical bioanalyzer profiles for RNA sequencing using Axon-seq. Axonal cDNA samples have a distinct profile compared to somatodendritic samples. An example of ready to sequence axonal sequencing library is also shown after tagmentation reaction and barcoding. Add a solution of 4% formaldehyde in PBS. When adding the formaldehyde, maintain similar volumes in all wells as used during media changes (see Figure 2) to ensure a flow both through the compartments and across the microgrooves. Store devices in PBS (~200 μl in all wells) until further use. If not used within a few days, wrap the 10 cm dish with parafilm.Note: Either before or after performing the desired staining method (immunocytochemistry/FISH/RNAscope), devices need to be removed along with coverslips from the 10 cm plastic dish. Using a 1 ml pipette, add a thin line of H2O around the edges of the cover glass. This will loosen it from the underlying surface and cause it to float after a few seconds/minutes. After RNA sequencing has been performed, a bioinformatic quality control (QC) step should be performed to exclude axonal samples with (traces of) soma contamination.Note: The following is a simplified version of the bioinformatic QC that is conducted using the programming language R (R Core Team, 2018). More in-depth knowledge of bioinformatics and R will be required to perform these analyses, but what follows is a general guide to exclude any soma-contaminated samples. Ensure that the RNA sequencing data is processed to both a count table (raw read counts) and an RPKM table with normalized values (reads per kilobase per million mapped reads). As a general QC guideline, samples with too few mapped reads or detected genes should be excluded. To perform this, extract the total number of counts (= mapped reads) per sample from the count table, as well as the total number of genes with counts. After this general QC, the data can be inspected for soma-contamination of axonal samples. Briefly, there are four indicators to investigate:The number of detected genes in axonal samples2D visualization plots (PCA/umap)Sample correlations in a correlation heatmapHierarchical clustering based on all expressed genes (or a subset) Obtain the number of detected genes in the axonal samples, and plot these as a dot plot. As a rough guideline, axon samples contain fewer than ~8,000 genes. Investigate any outliers in this distribution. Soma-contaminated samples will have a strong increase in the number of detected genes. Using the base R function prcomp, or alternatively the package umap (Konopka, 2018), plot a 2D visualization of your samples (first 2 principal components for prcomp, or n_components = 2 for umap). Investigate the plot. Typically, axon and soma samples will separate strongly in 2D space. Contaminated axon samples will be significantly shifted towards the somatic samples (Figure 5). Using the base R function corr, calculate the correlation between samples based on all expressed genes. This correlation matrix can be plotted as a heatmap using the function pheatmap from the package carrying the same name. Typically, axonal samples have a low correlation to somatic samples. However, contaminated samples will have an increased overall correlation to all somatic samples. Using pheatmap (Kolde, 2019), a heatmap with corresponding hierarchical tree clustering can be generated based on all expressed genes in the dataset. Where axon and soma samples normally cluster in distinct branches, contaminated samples can cluster together with soma samples.Figure 5. Example PCA-plot showing the shift of contaminated axon samples towards the somatic samples. They additionally have an increase in the number of detected genes per sample. Using these four measures, one can obtain an idea of samples that are possibly soma contaminated. Note that cutoffs need to be set for each of the measures, and the decision to exclude samples depends on the severity of the contamination.240 ml DMEM/F12-GlutaMAX10 ml B27 supplement, custom (stock 50x, add the full supplement bottle)5 ml Penicillin/Streptomycin5 ml GlutaMAX supplement0.91 ml 2-Mercaptoethanol1 bottle (500 ml) of DMEM/F12-GlutaMax5 ml N2 supplement (stock 100x, add the full supplement bottle)5 ml Penicillin/Streptomycin1 bottle (500 ml) of Neurobasal10 ml B27 supplement, custom (stock 50x, add the full supplement bottle)5 ml Penicillin/Streptomycin Lysis/harvesting solution (~70 μl per microfluidic device)2% Triton X-100 in nuclease-free dH2O. Cool on ice, then supplement with:0.5 μl RNase-inhibitor per 100 μl (final concentration of 0.2 U/μl)1 mM dithiothreitol (DTT), required for RNase-inhibitor enzymatic activity This work was supported by grants from the Swedish Research Council [2016-02112 and 2018-03255]; EU Joint Programme for Neurodegenerative Disease (JPND) [529-2014-7500]; the Strategic Research Programme in Neuroscience (StratNeuro); Karolinska Institutet; Birgit Backmark’s Donation to ALS Research at Karolinska Institutet in memory of Nils and Hans Backmark; Åhlén-stiftelsen (mA1/h17 and mA1/h18); Ulla-Carin Lindquists stiftelse för ALS forskning; and Magnus Bergvalls stiftelse [2015-00783 and 2016-01531] to E.H. J.A.B. is supported by a postdoctoral fellowship from the Swedish Society for Medical Research (SSMF). This protocol is based on our previous study published in Stem Cell Reports in 2018: Nijssen et al. (2018). Competing interests The authors declare that there are no financial or non-financial competing interests. Ethics All work was carried out according to the Code of Ethics of the World Medical Association (Declaration of Helsinki) and with national legislation and institutional guidelines. The use of human stem cell lines was approved by the regional ethical review board in Stockholm, Sweden (Regionala Etikprövningsnämnden, Stockholm, EPN). References Briese, M., Saal, L., Appenzeller, S., Moradi, M., Baluapuri, A. and Sendtner, M. (2016). Whole transcriptome profiling reveals the RNA content of motor axons. Nucleic Acids Res 44(4): e33. Konopka T. (2018). R-package: umap. Uniform Manifold Approximation and Projection. Nichterwitz, S., Chen, G., Aguila Benitez, J., Yilmaz, M., Storvall, H., Cao, M., Sandberg, R., Deng, Q. and Hedlund, E. (2016). Laser capture microscopy coupled with Smart-seq2 for precise spatial transcriptomic profiling. Nat Commun 7: 12139. Nichterwitz, S., Benitez, J. A., Hoogstraaten, R., Deng, Q. and Hedlund, E. (2018). LCM-Seq: A Method for Spatial Transcriptomic Profiling Using Laser Capture Microdissection Coupled with PolyA-Based RNA Sequencing. Methods Mol Biol 1649: 95-110. Nijssen, J., Aguila, J., Hoogstraaten, R., Kee, N. and Hedlund, E. (2018). Axon-Seq Decodes the Motor Axon Transcriptome and Its Modulation in Response to ALS. Stem Cell Reports 11(6): 1565-1578. R Core Team. (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. Rotem, N., Magen, I., Ionescu, A., Gershoni-Emek, N., Altman, T., Costa, C. J., Gradus, T., Pasmanik-Chor, M., Willis, D. E., Ben-Dov, I. Z., Hornstein, E. and Perlson, E. (2017). ALS along the axons–expression of coding and noncoding RNA differs in axons of ALS models. Sci Rep 7: 44500. Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W. and Jeon, N. L. (2005). A microfluidic culture platform for CNS axonal injury, regeneration and transport. Nat Methods 2(8): 599-605. 有一种不同于细胞体的RNA组成。因此，要充分了解健康和疾病中的神经元生物学我们需要研究躯体，树突和轴突。我们在这里描述一种新的方法的详细方案 我们还详细介绍了如何从小鼠和人类多能干细胞产生运动神经元进行测序，但Axon-seq适用于seq，用于轴突（和树突）的RNA测序。 仔细的生物信息学步骤确保容易识别和去除任何受体细胞污染的样品.-然后，通过生长因子梯度募集轴突，裂解并直接加工成不经RNA分离的cDNA。【背景】神经元是高度极化的细胞，它们的过程，包括树突和轴突，需要能够以独立于体细胞的方式响应微环境的变化（Holt和Schuman，2013）。这对于神经元特别重要，其中轴突构成细胞体积的主要成分，如脊柱运动运动的情况。为了分离神经突，有很好的工具，如Campenot Chambers（Boyden，1962; Campenot，1977）或微流体室（Taylor et al。， 2005）。如果神经突在这些装置中被分离出来，重要的是要记住隔室之间的交叉污染仍然可能发生。因此，孤立的轴突的RNA测序（Minis et al。，2014。 Saal et al 。，2014; Briese et al。，2016; Rotem et al。。，2017）可能导致错误的结果/结论这是对肌动蛋白mRNA组成及其在ALS中的调节的详细分析和准确研究，我们开发了Axon-seq（Nijssen et al。，2018）。我们的LCM-seq方法在单细胞空间RNA测序中的应用（Nichterwitz 等。，2016）。由于运动神经元具有轴突的神经，所述轴突具有纵向距离，所述轴突可用于流体装置以将来自干细胞的运动神经元（小鼠和人）的轴突与其胞体分离。这对于所有神经元亚型可能是不可能的，但该方法仍然允许将神经突分析为实体。与以前的方法相比，Axon-seq不需要RNA分离步骤，并且它允许从单个微流体装置进行高灵敏度和成本有效的测序。值得注意的是，Axon-seq有效地消除了所有具有任何体细胞交叉污染的样品，因为它使用了高严格性和敏感性的生物信息质量控制步骤Axonal样品含有来自未获得的背景的mRNA的可追溯性水平-seq可以应用于包含更长进程的任何单元格。

关键字：RNA测序, 运动神经元, 轴突, 干细胞, 微流控装置, 生物信息学

软铅笔型刷（任何软刷类型都没有太强的毛发，可能刺穿/损坏PDMS表面，其中制造微流体设备。刷子，来自典型的硬件/便利店，直径为2 5毫米工作效果最好，不需要实验室级别。尼龙/合成毛发不是问题。） 抗坏血酸，100mg粉末（Sigma-Aldrich，目录号：A4403），通过将粉末溶解在2.84ml无核酸酶的dH O O中制备200mM储备溶液。 Y-27632,10mg粉末（Tocris，目录号：1254），通过将粉末溶解在3.12ml无核酸酶的dH 2 O中制备成10mM储备溶液。 LDN-193189二盐酸盐，10mg粉末（Tocris，目录号：6053），通过将粉末溶解在2.09ml DMSO中制备成10mM储备溶液，然后在DMSO中制备1mM工作原液（1:10稀释） SB-431542,10mg粉末（Tocris，目录号：1614），通过将粉末溶解在2.60ml DMSO中制备成10mM储备溶液 CHIR-99021,10mg粉末（Tocris，目录号：4423），通过将粉末溶解在7.16ml DMSO中制备成3mM储备溶液 DAPT，10mg粉末（Tocris，目录号：2634），通过将粉末溶解在2.31ml DMSO中制备成10mM储备溶液 SAG，1mg粉末（Tocris，目录号：4366），通过将粉末溶解在4.08ml DMSO中制备成500μM储备溶液 全部反式视黄酸，50mg粉末（Sigma-Aldrich，目录号：R2625），通过将粉末溶解在1.66ml DMSO中制备100mM储备溶液，然后制备1mM工作原液（1 ：在DMSO中稀释100倍 胶质衍生的神经营养因子（GDNF），10μg粉末（Peprotech，目录号：450-10），通过将粉末溶解在含有0.1％BSA的1ml dPBS中制备成10μg/ ml储备溶液 脑源性神经营养因子（BDNF），10μg粉末（Peprotech，目录号：450-02），通过将粉末溶解在含有0.1％BSA的1ml dPBS中制备成10μg/ ml储备溶液 微流体装置的储存和清洗来自Xona MicrofluidicsTM（标准神经元器件）的具有任何沟槽长度的商业微流体装置可以存储在dH 2 O中并且多次使用。注意：器械的沟槽长度可能会影响轴突与树突的比例，这些比例可以穿过相对的腔室，也可能影响器械中培养时间的长度。对于小鼠神经元，我们使用150个器械对于人类运动轴突，我们使用具有450μm沟槽的装置。长度为0.3μm的沟槽并且在培养的7天期间看到大部分轴突被回收。 将装置置于具有500ml 1％Triton X-100的dH 2 O的玻璃烧杯中2小时，并使用磁力搅拌器保持装置搅拌。 每次在新鲜的dH 2 O中洗涤3×10分钟以除去Triton X-100的任何残留物。然后，在70％乙醇中的无菌装置至少10分钟。注意：洗涤程序可以随时进行，设备可以在70％乙醇中储存，以备不时之需。在涂层程序启动当天启动设备附件（下图）。 使用无菌镊子从70％乙醇中取出微流体装置，让装置在无菌层流罩中风干20分钟。使用吸气器除去残留的乙醇。注意：在此阶段设备完全干燥非常重要，因为任何剩余的液体都会对设备连接产生负面影响，并可能导致后期泄漏。 将无菌玻璃盖玻璃（32 x 24 mm，厚度＃1或＃1.5）放在水滴顶部，以确保盖玻片不会在餐具内移动（图1B）。 使用设备背面（凹槽上方）（凹槽上方）（凹槽上方）轻轻施力如果所有凹槽位于同一焦平面并因此连接到盖子上，则标记可用作驴子的主题。以这种方式连接的设备至少稳定四周。细胞将在一个角落附着防水标记物附着在玻璃表面上，以确定装置未来的左右方向。该装置形成细胞的\"天花板”，并为轴突产生凹槽。盖玻片。 设备也可以直接放置在组织培养级塑料盘中（即，直接放在塑料上，没有盖玻片）。设备的最终设置（步骤B4）。然而，成像目的，将它们放在玻璃盖玻片上是有益的。 ＆nbsp;除了此处描述的PDMS设备外，Xona Microfluidics TM 还提供用于细胞培养的预粘合设备（XonaChips）。虽然这些设备与普通设备相同，但它们不是最适合重复使用，可以使用常规设备（SND150）。对于预粘合设备，可以跳过附加步骤（程序B）。 图1.在10 cm培养皿中设置4个微流体装置。 A.首先，将4滴4μldH 2 O均匀分布在10 cm培养皿中。 B.安装盖玻片时，将一个微电流装置的特征面朝下放在每个盖玻片上，轻轻按下滴液玻璃盖玻璃的顶部，在盖玻片和塑料盘之间形成水封。 C.在一个10厘米的盘子中最终设置四个设备。如果需要，可以进行其他设备布置（就餐具尺寸而言）。＆nbsp; 如果没有发生这种情况，请使用带有1 ml移液器吸头的吸气器，将液体吸入每个装置的两个底部孔中。顶部。不要吸入所有液体。 使用1x PBS洗涤设备两次。洗涤，从孔中吸出涂层，然后将PBS加入孔中，底部75μl，然后顶部150μl。 第二层涂层由2μg/ ml纤连蛋白和5μg/ ml层粘连蛋白在PBS中的溶液组成。以与第一层相似的方式添加最后一层涂层 - 参见步骤C2-C4。 小鼠胚胎干细胞运动神经元的规格通常，分化的良好起点是干细胞的10cm培养皿，融合度为约70-80％。细胞总是保持在37℃和5％CO 2 的潮湿培养箱中。 br />第1天： 预热的TrypLE Express用于在37°C（即，2 ml用于6 cm培养皿，4 ml用于10 cm培养皿）中在管中解离。此外，预热管8ml DMEM / F12-GlutaMAX（或任何其他基于MEM的普通介质）。 当细胞倒圆并从培养皿中取出时，使用移液管将它们完全分离在TrypLE Express中，然后将所有物质转移到预热的DMEM管中。 将细胞置于细菌（非粘附）培养皿中，密度为0.5至1.5百万个细胞/ ml（细胞数量依赖于细胞系，需要进行测试），并以30 rpm的速度放置在低速轨道振荡器上在孵化器中过夜。 要更换EB上的培养基，将整个培养基（包括EB）移液到管中。让EB下沉约2分钟并除去上清液。重新吸收EBs将1ml新鲜培养基放入新培养皿中。 更换为新鲜小鼠MN培养基，补充100 nM全 - 反式 - 维甲酸（RA）和500 nM平滑激动剂（SAG）。重复4天，见表1. 表1.小鼠多能干细胞分化运动神经元的因素 来自人多能干细胞的运动神经元的规格通常，分化的良好起点是10 cm的干细胞培养皿，融合度约为70-80％。细胞总是保持在37°C和5％CO 2的加湿培养箱中。 第1天： 然而，最后，以0.5M-0.75M细胞/ ml的密度重悬细胞在人EB培养基中（该密度可以与EB日相同）。补充第1天人类EB媒体，其中包含第1天所需的因素（概述见表2）：5μMY-27632（ROCK抑制剂）200 nM LDN-193189 40μMSB-431542 3μMCHIR-99021 200μM抗坏血酸表2.运动神经元从人多能干细胞分化的因素 第2-10天： 每隔约24小时进行一次每日培养基更换，并记录每天的相应因素（表2）。注意：虽然协议的RA-SAG阶段（第3-10天），但并非绝对要求媒体更改遵循24小时间隔，并且必要时（但不推荐）可以省略一天的媒体更改。但是，至关重要的是，前两天的双SMAD抑制（使用SB-431542和LDN-193189）是48小时而不是更短。第10天： 细胞准备分离成种子流体装置（见下文）。 将1ml预热（37°C）TrypLE Express加入含有EB的Falcon试管中，解离需要10-20分钟，具体取决于EB的大小。 每隔5分钟轻轻搅动EB，方法是轻拍管或用1 ml移液管轻轻上下移液。注意：如果您在TrypLE Express中5分钟后第一次用EB暂停，请不要将它们吸移到移液器吸头中，因为它们位于内部内侧的尖端。在暂停中稍微激动。 加入9毫升预热的DMEM稀释1毫升的TrypLE Express。一旦EB开始分解，上下移液相当明显地大约10次以完成解离。 通过70μm细胞过滤器过滤器过滤所得悬浮液以除去未分布的团块/碎片，然后在室温下以200 x g 旋转4分钟。 将密度调节至每μl2.5×10 4个细胞的最终浓度（这将确保随后每个微流体装置接种总共1×10 5个细胞）将细胞重新悬浮在所需量的培养基中（即 ），将1ml细胞悬浮液转移到Eppendorf管中，再以200 xg 旋转4分钟。 >，每2.5 x 10 4个细胞1μl。在此阶段，细胞应重悬于最终培养基中，对于人细胞，这是具有第10天因子的B27培养基（参见表10）。 2），对于小鼠细胞，这是具有第7天因子的小鼠MN培养基（参见表1）。如何，强烈建议添加抗坏血酸和营养因子（以及人体细胞的ROCK抑制剂）以提高电镀后的存活率在微流体装置的受限环境中。 准备最后足够的介质以填充所有设备（每个设备0.5毫升）。最后，设备将保持介质卷，如图2所示。 加入营养因子使浓度增加到50 ng / ml。\"取所需量的培养基（见图2）”对轴突室（顶部和底部孔）转移到新管中的营养因子浓度越高。轴突\"侧面将改善整个微槽的轴突募集。 图2.播种时微量流体装置中的培养基体积和营养因子浓度。请注意，人类和小鼠MN之间的神经营养因子的体细胞浓度不同。轴突穿过微槽进入表面后对于小鼠和人来说，两侧的因子浓度均等于10ng / ml。 将4μl培养基（含有50 ng / ml BDNF / GDNF）加入轴突侧的腔室中。移液器靠近左上方的腔室入口（图2中的方向，见图3A中的照片）并允许资本力将液体吸入腔室。 一旦细胞出现附着在细胞上，细胞在检查细胞并附着在孔上后放置在装置上10分钟。孵化器过夜。注意： 如果发生这种情况，我们建议仅向所有孔中添加40μl培养基（每侧保持正确的培养基类型）偶尔细胞即使在15分钟后也不能很好地附着，可能部分是由于隔室之间的流动。这样，流量最小，蒸发也不会成为问题。然后将这样的单元格再保持一小时，最后将介质添加到图2中的推荐量。 除了此处描述的所有内容外，如表2所述，每天在培养基中保持相应的因子。营养因子（BDNF / GDNF）浓度 有一个体细胞室，轴向室在最初的几天内接受营养因子梯度（见下文）。 图3.将细胞接种到微流体室和代表性显微照片中。 A.可以将介质/细胞添加到微流体装置的腔室中的角度的示例照片。虚线表示腔室的位置在轴突室和体细胞室中的良好培养物的显微照片，其中可见神经元胞体簇。 将神经元接种到微流体装置后，每天更换培养基以确保营养因子梯度保持完整。可以使用明视野显微镜来跟踪轴突生长。注意：更换培养基时，不要将培养基从培养室中吸出，因为这样会正式清除所有细胞/轴突，只更换孔中的培养基。 一旦看到轴突穿过几个微槽进入轴突区室（即> 10个带轴突的凹槽），可以去除营养因子梯度，并且两个隔室都可以接收参见图3B，获得具有生长轴突的微流体装置的成功显微照片。 准备裂解/收获溶液。每个微流体装置准备~70μl溶液，或者好像只收集一个隔室（最终推荐的溶解体积为50μl用于体细胞室，10μl用于轴突补体）确保最终溶液中存在更高浓度的RNA，这有助于下游应用。 裂解溶液（参见食谱）由无核酸酶dH 2 O的2％Triton X-100溶液组成。首先，将该溶液在冰上冷却，然后补充：每100μl含0.5μlRNase抑制剂（终浓度0.2 U /μl）RNase抑制剂酶活性所需的1 mM二硫苏糖醇（DTT）注意：在准备和使用过程中，始终将裂解液保持在冰上。 如果在轴突样品的下游生物信息学分析中检测到体细胞污染，则可以针对任何交叉竞争的体细胞仔细检查这些图像。 将溶液转移到制备的Eppendorf管中并快速 - 等待10秒，然后在底部孔中上下移液~5x，并从室底部提取尽可能多的溶液（可以>10μl）。在干冰上冻结。 对于体细胞侧重复上述步骤I7和I8，但是使用50μl裂解液。当提取溶液时，尝试尽可能多地提取（>50μl）。在干冰上快速冷冻。注意：有关无RNA提取的RNA测序文库制备的详细说明，方案和试剂，请参见Nichterwitz等，2018。生成轴向和体细胞测序文库，标准实验室实践与成为相关使用经过RNAse / DNAse认证的一次性用品和经过分子生物学等级认证的试剂。考虑专用工作站（工作台）和代表隐私的热循环仪，即保持样品冷（4°C）和表面清洁。由于每个设备的材料（RNA）数量少，与单个细胞的材料相容，我们采用了Smart-seq2协议，这是一种用于单细胞转录组学的既定方法。简要说明，样本很快从第一步开始使用oligo-dT引物和模板开关LNA-oligo使-80℃和10μl轴突裂解物（5μl体树突裂解物）进行逆转录。此时，cDNA的质量和数量可以通过生物分析仪使用dsDNA高灵敏度来测量。最后，标记了cDNA文库（Nextera XT Kit，Illumina）和带有Illumina指数的条形码，用于RNA测序。 图4.使用Axon-seq进行RNA测序的典型生物分析仪图。轴突cDNA样品与体细胞树突样品相比具有明显的特征。 添加甲醛时，在介质更换过程中使用的所有孔中保持相似的体积（参见图2）。随后的流体通过隔室和微槽。 将设备储存在PBS中（所有孔中约200μl）直至进一步使用。如果在几天内未使用，请用封口膜包裹10 cm培养皿。注意：在进行所需的染色方法（免疫细胞化学/ FISH / RNAscope）之前，需要将设备与10cm塑料盘中的盖玻片一起移除。 使用1毫升移液器，在盖玻片的边缘周围添加一条细线H 2 O.这将失去表面并使其在几秒/分钟后漂浮。 在进行RNA测序后，应进行生物信息学质量控制（QC）步骤以排除具有（痕量）体细胞污染的轴突样品。注意：以下是使用编程语言R（R核心团队，2018）进行的生物信息学QC的简化版本。更深入的生物信息学知识和R以下是排除任何受躯体污染的样本的一般指南。 确保将RNA测序数据处理为计数表（原始读取计数）和具有标准化值的RPKM表（每千位基因读数的每千碱基读数）。 为了执行此操作，应从计数表中提取每个样本的总计数（=映射读数），以及排除具有太少的映射读数或检测到的基因的样本的基因总数。计数。 在这个一般的QC之后，可以检查数据是否有轴突样本的躯体污染。简而言之，有四个指标需要调查：轴突样本中检测到的基因数量2D可视化图（PCA / umap）相关热图中的样本相关性基于所有表达基因（或子集）的分层聚类 受到体细胞污染的样本将在轴突样本中进行，绘图将作为点图。作为粗略的指导线，轴突样本包含超过8,000个基因。调查此分布中的任何异常值。检测到的基因数量。 使用基本R函数 corr ，基于所有表达的基因计算样本之间的相关性。可以使用来自包含所有表达基因的函数 pheatmap 将该相关矩阵绘制为热图。通常，轴突样本与体细胞样本具有低相关性。然而，争用样本将与所有体细胞样本具有增加的总体相关性。 在 pheatmap （Kolde，2019）中，可以基于数据集中的所有表达基因生成具有相应分层树聚类的热图。其中聚类通常被划分为不同的聚类，区分聚类以分析样本用soma样品。 图5.示例PCA图显示污染样品向体细胞样品的移动。它们还增加了每个样品检测到的基因数量。 请注意，需要为每个测量设置截止值，排除样品的决定取决于污染的严重程度。使用这四个测量，人们可以了解可能受到污染的样品。 裂解/收获溶液（每个微流体装置约70μl）2％Triton X-100无核酸酶dH 2 O.在冰上冷却，然后补充：每100μl含0.5μlRNase抑制剂（终浓度0.2 U /μl）RNase抑制剂酶活性所需的1 mM二硫苏糖醇（DTT） 欧盟神经退行性疾病联合计划（JPND）[529-2014-7500];神经科学战略研究计划（StratNeuro）;卡罗林斯卡医学院;Ahlén-Stiftelsen（mA1 / h17和mA1 / h18）; Ulla-Carin Lindquists支持ALS forskning;和Magnus Bergvalls stiftelse [2015-00783和2016] -01531]到EHJAB获得瑞典医学研究学会（SSMF）的博士后奖学金。＆nbsp;该协议基于我们之前在2018年的干细胞报告中发表的研究：Nijssen et al。（2018）。竞争利益作者声明没有财务或非财务竞争利益。 瑞典斯德哥尔摩区域伦理审查委员会批准使用人类干细胞系（区域） Etikprövningsnämnden，斯德哥尔摩，EPN）。 Boyden，S。（1962）。抗体和抗原混合物对多态性白细胞的趋化作用。 J Exp Med 115：453-466。 Briese，M.，Saal，L.，Appenzeller，S.，Moradi，M.，Baluapuri，A。和Sendtner，M。（2016）。整个转录组分析可以恢复运动轴突的RNA含量。 Nucleic Acids Res 44（4）：e33。 Campenot，RB（1977）。通过神经生长因子局部控制神经元发育。 Proc Natl Acad Sci USA 74（10）：4516-4519。 Holt，CE和Schuman，EM（2013）。分散的中心法则：关于RNA功能的新观点神经元中的局部翻译。 Neuron 80（3）：648-657。 Nichterwitz，S.，Chen，G.，Aguila Benitez，J.，Yilmaz，M.，Storvall，H.，Cao，M.，Sandberg，R.，Deng，Q。和Hedlund，E。（2016）。 a href =\"http://www.ncbi.nlm.nih.gov/pubmed/27387371”target =\"_ blank”>激光捕获显微镜结合Smart-seq2进行精确的空间转录分析。 Nat Commun 7：12139。 Nichterwitz，S.，Benitez，JA，Hoogstraaten，R.，Deng，Q。和Hedlund，E。（2018）。 LCM-Seq：使用激光捕获显微切割与基于PolyA的RNA测序偶联的空间转录组分析方法。 方法Mol Biol 1649：95-110。 Nijssen，J.，Aguila，J.，Hoogstraaten，R.，Kee，N。和Hedlund，E。（2018）。 Axon-Seq解码运动轴突转录组及其响应ALS的调节。 干细胞报告 11（6）：1565-1578。 R核心团队。（2018）.R：统计计算的语言和环境.R统计计算基金会，奥地利维也纳。 https://www.R-project.org/。 Rotem，N.，Magen，I.，Ionescu，A.，Gershoni-Emek，N.，Altman，T.，Costa，CJ，Gradus，T.，Pasmanik-Chor，M.，Willis，DE，Ben-Dov ，IZ，Hornstein，E。和Perlson，E。（2017）。沿着轴突的ALS-在ALS模型的轴突中编码和非编码RNA差异的表达。 Sci Rep 7：44500。 Saal，L.，Briese，M.，Kneitz，S.，Glinka，M。和Sendtner，M。（2014）。脊髓性肌萎缩细胞培养模型中的亚细胞转录组改变指向轴突生长和突触前分化的广泛缺陷。 RNA 20（11）：1789-1802 。 Taylor，AM，Blurton-Jones，M.，Rhee，SW，Cribbs，DH，Cotman，CW and Jeon，NL（2005）。用于CNS轴突损伤，再生和转运的微流体培养平台。 Nat Methods 2（8）：599-605。 为了向广大用户提供经翻译的内容，www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高，也不及 100% 的人工翻译的质量。为此，我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。 引用：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. （提问前，请先登录）bio-protocol作为媒介平台，会将您的问题转发给作者，并将作者的回复发送至您的邮箱（在bio-protocol注册时所用的邮箱）。为了作者与用户间沟通流畅（作者能准确理解您所遇到的问题并给与正确的建议），我们鼓励用户用图片的形式来说明遇到的问题。当遇到任何问题时，强烈推荐您通过上传图片的形式提交相关数据。 I was diagnosed of ALS few years ago. I was given medications which helped but only for a short time. So i decided to try alternative measures and began on ALS HERBAL PROTOCOL from Herbal HealthPoint, It has made a tremendous difference for me (Go to ww w. herbalhealthpoint. c o m).  I had improved walking balance, increased appetite, muscle strength, improved vision. I totally recovered, I am symptom free