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Mar 2019

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Labeling and Isolation of Fluorouracil Tagged RNA by Cytosine Deaminase Expression
利用胞嘧啶脱氨酶表达进行氟尿嘧啶标记RNA标记和分离    

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Abstract

Tissues are comprised of different cell types whose interactions elicit distinct gene expression patterns that regulate tissue formation, regeneration, homeostasis and repair. Analysis of these gene expression patterns require methods that can capture as closely as possible the transcriptomes of cells of interest in their tissue microenvironment. Current technologies designed to study in situ transcriptomics are limited by their low sensitivity that require cell types to represent more than 1% of the total tissue, making it challenging to transcriptionally profile rare cell populations rapidly isolated from their native microenvironment. To address this problem, we developed fluorouracil-tagged RNA sequencing (Flura-seq) that utilizes cytosine deaminase (CD) to convert the non-natural pyrimidine fluorocytosine to fluorouracil. Expression of S. cerevisiae CD and exposure to fluorocytosine generates fluorouracil and metabolically labels newly synthesized RNAs specifically in cells of interest. Fluorouracil-tagged RNAs can then be immunopurified and used for downstream analysis. Here, we describe the detailed protocol to perform Flura-seq both in vitro and in vivo. The robustness, simplicity and lack of toxicity of Flura-seq make this tool broadly applicable to many studies in developmental, regenerative, and cancer biology.

Keywords: In situ transcriptomics (原位转录组学), Cytosine deaminase (胞嘧啶脱氨酶), Uracil phosphoribosyl transferase (尿嘧啶磷酸核糖转移酶), Fluorouracil (氟尿嘧啶), Nascent RNA (新生RNA)

Background

In an organ, cells interact with tissue microenvironment that includes organ-specific resident cells, immune cells, perivascular niches, extracellular matrix, cytokines, metabolites, and an oxygen concentration range. These interactions dictate the expression of specific set of genes in the cells that often have functional significances. The transcriptional responses associated with these interactions are usually dynamic and can only be observed if the interactions are preserved. Thus, it is critical to maintain the tissue microenvironment to ensure the capture of true transcriptional state of cells under the physiological conditions.

Current techniques to study cell-type specific transcriptomes have limitations that preclude their effective application in studying rare and under-represented cell populations. Single-cell RNA sequencing (scRNA-seq) with or without an intervening fluorescence activated cell sorting (FACS) step, requires extensive physical and enzymatic processing of the tissue, which not only disrupts the effects of the host microenvironment on cells but also exerts stress on these cells, severely compromising the ability to discern the impact of the host stroma from the transcriptome of the isolated cells. As many as 7,500 genes have been reported to be altered by more than two-fold during the FACS processing of muscle stem cells for transcriptomic analysis (Machado et al., 2017). In situ transcriptomic profiling obviate these problems but lack the necessary sensitivity for cell populations that represent less than 1% of the tissue. For example, translating ribosome affinity purification and mRNA sequencing (TRAP-Seq) (Heiman et al., 2008) is not suitable to analyze cells that constitute less than 1% of the total population (Bertin et al., 2015; Obenauf et al., 2015). Direct enzyme based metabolic tagging of RNA with thiouracil (TU) and ethynyl cytosine (EC) in the cells of interest are limited in sensitivity and specificity due to collateral tagging of RNA in cells lacking the enzymes, and requires additional in vitro biotinylation steps (Cleary et al., 2005; Miller et al., 2009; Gay et al., 2013 and 2014; Hida et al., 2017). TU tagging has a sensitivity limit of 5% (Gay et al., 2013). Thiol (SH)-linked alkylation of the metabolic labeling of RNA in tissue (SLAM-ITseq) eliminates the noise associated with the purification of RNAs that are not thiol tagged in TU-tagging method (Matsushima et al., 2018), but undesired TU tagging through endogenous enzymes in cells lacking UPRT expression remains a limitation. We described the development of Flura-seq, a cytosine deaminase (CD)-based method for in situ transcriptomic profiling of rare cell populations that represent as little as 0.003% of an organ (Basnet et al., 2019). Flura-seq requires exogenous expression of CD, a key enzyme of the pyrimidine salvage pathway in fungi and prokaryotes (Mullen et al., 1992) and UPRT from T. gondii. CD is absent in mammalian cells, which instead use cytidine deaminase for the same purpose (Mullen et al., 1992). In addition to converting cytosine to uracil, CD can also convert 5-fluorocytosine (5-FC), a non-natural pyrimidine, to 5-flourouracil (5-FU). 5-FU is endogenously converted to fluorouridine triphosphate (F-UTP), which is then incorporated into RNA. The co-expression of UPRT limits the labeling of RNA in the cells expressing CD (Basnet et al., 2019). Flura-seq is applicable to in vitro co-culture experiments as well as in vivo experiments where cells of interest, expressing CD-UPRT, are present in the intact tissue. Flura-seq is technically very simple that only requires very basic molecular biology skills, and the whole procedures can be completed within 24 hours. Here, we describe the detailed protocol for labeling and isolation of RNA for Flura-seq for both in vitro and in vivo experiments.

Materials and Reagents

  1. 5 ml Polystyrene Round-Bottom tube (Corning, catalog number: 352058)
  2. 60 mm Tissue culture plates (Corning, catalog number: 353004)
  3. Oligo (dT)25 magnetic beads (New England Biolabs, catalog number: S1419S)
  4. Protein G Dynabeads (Thermo Fisher Scientific, catalog number: 10004D)
  5. 6-8 weeks old athymic nude female mice (Envigo, catalog number: 069)
  6. Doxycycline diet (625 mg/kg, Envigo, catalog number: TD.07383)
  7. BrdU antibody (Abcam, catalog number: ab6326)
  8. CD-UPRT (Addgene, plasmid number: 126677)
  9. rtTA3 (Addgene, plasmid number: 26730)
  10. Doxycycline (Sigma-Aldrich, catalog number: D9891)
  11. 5-Fluorocytosine (5-FC) (Sigma-Aldrich, catalog number: F7129)
  12. 5-Bromo-2’-deoxyuridine (BrdU) (Millipore Sigma, catalog number: B5002)
  13. Thymine (Sigma-Aldrich, catalog number: T0376)
  14. RNeasy MinElute Cleanup kit (Qiagen, catalog number: 74204)
  15. cDNA kit-First Strand Transcriptor (Roche, catalog number: 043790-12001)
  16. RNeasy Mini Kit (Qiagen, catalog number: 74106)
  17. Ethylenediaminetetraacetic acid solution (EDTA) (Sigma-Aldrich, catalog number: 03690)
  18. Tween 20 (Sigma Millipore, catalog number: P1379)
  19. Lithium dodecyl sulfate (LiDS) (Sigma Millipore, catalog number: L4632)
  20. Lithium chloride (LiCl) (Sigma Millipore, catalog number: 203637)
  21. Ultrapure BSA (BSA) (Thermo Fisher Scientific, catalog number: AM2616)
  22. Glycogen (Thermo Fisher Scientific, catalog number: R0551)
  23. RNAlater (Sigma-Aldrich, catalog number: R0901)
  24. 20x UltraPure SSPE Buffer (Thermo Fisher Scientific, catalog number: 15591043)
  25. PBS
  26. Tris base
  27. Tris-HCl
  28. Ethanol (Decon Labs, catalog number: 2716)
  29. DTT (Sigma-Aldrich, catalog number: 3483-12-3)
  30. Cell lysis buffer (see Recipes)
  31. mRNA Wash Buffer I (see Recipes)
  32. mRNA Wash Buffer II (see Recipes)
  33. mRNA Wash Buffer III (see Recipes)
  34. Elution Buffer (see Recipes)
  35. SSPET buffer (see Recipes)
  36. TE buffer (see Recipes)

Equipment

  1. PRO 200 grinder from PRO Scientific Inc (Homogenizer) (PRO Scientific, catalog number: 01-01200) 
  2. Magnetic rack (ThermoFisher Scientific, catalog number: 12321D)
  3. Thermo scientific Rotator (ThermoFisher Scientific, catalog number: 88881001)
  4. Eppendorf Thermomixer (ThermoFisher Scientific, catalog number: 05-400-200)
  5. Centrifuge

Procedure

  1. Making CD-UPRT expressing stable cells
    1. Generate CD-UPRT expressing cell line of interest (for example, MDA-MB-231) by transducing cells with CD-UPRT (Addgene) and rtTA3 (Addgene) lentivirus using standard protocol.

  2. Labeling of RNA in vitro
    Day 1
    1. Plate 1,000 cells (for example, MDA-MB-231) expressing Doxycycline inducible CD-UPRT (cells of interest) with 1 million 4T1 cells (or other cells (optional)) in 60 mm plates in 3 ml volume (Doxycycline inducible system is optional).
    Day 2
    1. Add 1 μg/ml Doxycycline.
    Day 3
    1. 24 h post Doxycycline treatment, add 5-FC and thymine to 250 μM (50 mM stock) and 125 μM (25 mM stock) final concentration, respectively. 5-FC and thymine stock solutions are made in PBS. 
    2. Harvest cells after 2-12 h using cell lysis buffer. Wash the cells 1x with 3 ml PBS before adding 1 ml of cell lysis buffer on the plate. Scrape the cells in 1.5 ml eppendorf tube.

  3. Labeling of RNA in mice
    Day 1
    1. Prepare 500,000/ml CD-UPRT expressing stable cells (described above in A.1) in PBS.
    2. Inject 100 μl (50,000 cells) in mouse through tail vein.
    Day 28
    1. Change the mice diet to Doxycycline diet, 3 days before 5-FC injection, if cells are expressing Doxycycline inducible CD-UPRT. Skip this step if cells are constitutively expressing CD-UPRT.
    Day 31
    1. Inject the mice with 250 mg/kg 5-FC in PBS (12.5 μg/ml stock) intraperitoneally, and 125 mg/kg thymine in PBS (6.25 μg/ml stock) subcutaneously.
    2. After 4-12 h, euthanize the mice by CO2 asphyxiation, harvest the organs, and keep the organ in RNAlater buffer.
    3. Put the organ of interest (for example, lungs) in 2 ml cell lysis buffer (Recipe 1) in 5 ml Polystyrene Round-Bottom tube, and homogenize the organ with a tissue grinder.
    4. Divide the lysate equally into two tubes and add 3 ml of cell lysis buffer in each tube (The lysate can be flash frozen in liquid nitrogen for future use).

  4.  Isolation of mRNA
    1. Add 1x volume of cell lysis buffer (4 ml) in the lysate from C.7, rotate at room temperature (RT) for 5 min. For in vitro cells, add appropriate volume of lysis buffer (1 ml for confluent 60 mm plates), and scrape the cells.
    2. Put the lysate into 1.5 ml Eppendorf tube/s, and centrifuge at 16, 000 x g for 5 min at RT, and keep supernatant.
    3. Prepare oligo dT25 beads (≥ 100 μl beads/~25 mg of lungs/brain).
      1. Place 250 μl beads/Eppendorf tube (1.5 ml) in magnetic rack.
      2. Remove the storage buffer.
      3. Wash the beads 1x with 1 ml cell lysis buffer by pipetting up and down five times.
    4. Take out the supernatant (1 ml/250 μl beads) from D.2 and add it to the freshly prepared oligo dT25 beads.
    5. Incubate at RT for 10 min in a rotator.
    6. Discard the supernatant.
    7.  Wash the beads 2x with 1 ml mRNA wash buffer I (Recipe 2) at RT by pipetting up and down seven times.
    8. Wash the beads 2x with 1 ml mRNA wash buffer II (Recipe 3) at RT by pipetting up and down seven times.
    9. Wash the beads 1x with 1 ml mRNA wash buffer III (Recipe 4) at RT by pipetting up and down seven times.
    10. Wash the beads 1x with 1 ml TE buffer (Recipe 7) at RT by pipetting up and down seven times.
    11. Add 50 μl of Elution buffer (Recipe 5), and elute the RNA in Eppendorf Thermomixer at 85 °C for 2 min shaking at 750 rpm.
    12. Place the beads in magnetic rack and keep the Eluate. The mRNA can be stored at -80 °C for future use.

  5. Purification of 5-FU tagged mRNA
    1. Prepare 70 μl of protein G beads per sample at RT. Place the beads in magnetic rack, and remove the storage buffer. Upto 350 μl (for 5 samples) of beads can be transferred to one 1.5 ml Eppendorf tube.
    2. Wash the beads with 1 ml of 0.5x SSPET buffer (Recipe 6) at RT by pipetting up and down five times.
    3. Block the beads with 1 ml of 0.5x SSPET buffer containing 10 μg/ml BSA and 20 μg/ml glycogen for 1 h at 4 °C.
    4.  Wash the beads 1x with 1 ml of 0.5x SSPET buffer at RT by pipetting up and down five times.
    5. Add 35 μl of beads in 750 μl of 0.5x SSPET buffer containing BrdU antibody (1 μg for RT-PCR, and 5 μg for RNA-seq) in 1.5 ml Eppendorf tube. Rotate at 4 °C overnight.
    6. Wash the beads 1x with 1 ml of 0.5x SSPET buffer at RT by pipetting up and down five times.
    7. Add 35 μl of beads in mRNA (400 μl in this case) from Step D12 diluted with the same volume (400 μl) of 1x SSPET buffer. Total volume will be about 835 μl.
    8. Rotate for 2 h using ThermoScientific Rotator at RT.
    9. Wash 2x with 1 ml of 0.5x SSPET buffer at RT by pipetting up and down seven times.
    10. Wash 2x with 1 ml of 1x SSPET buffer at RT by pipetting up and down seven times.
    11. Wash 1x with 1 ml of TE buffer at RT by pipetting up and down seven times.
    12. Elute the bound mRNA with 200 μl of 100 μg/ml BrdU in TE buffer in Eppendorf Thermomixer at 900 rpm for 45 min at RT.
    13. Keep the supernatant (5-FU tagged mRNA).
    14. Mix the 200 μl supernatant with 700 μl of RLT buffer from RNeasy kit.
    15. Add 500 μl of 100% ethanol, and mix well.
    16. Pass the solution through RNeasy column.
    17. Wash 1x with 500 μl of RPE buffer from RNeasy kit.
    18. Wash 1x with 500 μl of 80% ethanol.
    19. Spin at 13,000 x g for 2 min at RT.
    20. Elute the RNA with 200 μl of RNase free water.
    21. Add 600 μl of 0.5x SSPET to the elute.
    22. Wash the Protein G Dynabeads incubated with BrdU antibody as described in Step E6.
    23. Re-immunoprecipitate 5-FU tagged mRNA as described in Steps E7-E13.
    24. Purify RNA as described in Steps E14-E19 using RNeasy MiniElute Cleanup kit, and elute the RNA with 12.5 μl RNase free water.
    25. Proceed with cDNA synthesis for RT-PCR or RNA-seq.

  6. Labeling and Purification of 5-Fluorouracil (5-FU) tagged mRNA from co-cultured cells and from mouse lungs in xenograft model
    1,000 human MDA-MB-231 cells transduced with CD-UPRT (Addgene) and rtTA3 (Addgene) (MDA231-CDUPRT) were co-cultured with 1 million mouse 4T1 cells. RNAs were labeled as described in Section B, and 5-FU tagged mRNAs were purified as described in Section D and E. Enrichment of 5-FU tagged mRNAs of representative human and mouse genes relative to non immuno-purified mRNA were measured by RT-PCR (Figure 1A).
      5 x 104 MDA231-CDUPRT cells were injected in the mouse lungs through tail vein. After 28 days, CD UPRT expression was induced by feeding mice with doxycycline diet for 3 days, and RNA was labeled as described in Section C, and 5-FU tagged mRNA was purified as described in Section D and E. Enrichment of 5-FU tagged mRNAs of representative human and mouse genes relative to non immuno-purified mRNA were measured by RT-PCR (Figure 1B).


    Figure 1. Labeling and isolation of 5-FU tagged RNA by cytosine deaminase expression. A. 1,000 human MDA-MB-231 cells expressing CD-UPRT were co-cultured with 1 million mouse 4T1 cells, and 5-FU tagged mRNA from human cells were isolated as described in the protocol, and enrichment of representative human genes and mouse genes relative to their corresponding 2% inputs (non immuno-purified mRNA) are shown (n = 3, ± SEM). B. 50,000 MDA-MB-231 cells expressing CD-UPRT were injected to the lungs through tail vein, and after 28 days, 5-FU tagged mRNA was isolated as described in the protocol, and the enrichment of representative human and mouse genes relative to 1% inputs (non immune-purified mRNAs) were measured by RT-PCR (n = 6, ± SEM). “h” indicates human and “m” indicates mouse.

Notes

  1. For in vitro experiments, confluency of cells does not affect 5-FU tagging (Step B1) and the method is likely to be applicable to all cell types (adherent as well as suspension cells). We have successfully applied the method in MDA-MB-231, HCC1954, H2087, 293T and 4T1 cells. 
  2. Thymine does not dissolve completely in PBS. Filter the solution before use (Step B3).
  3. Longer incubation of 5-FC and thymine increases labeling of RNA, and yields higher signal-to-noise ratio. 12 h of labeling is recommended for very small number of cells (< 0.01% or < 1,000 cells). For conditions consisting of more than 1 x 105 cells, 4 h of RNA labeling is sufficient. (Steps B4 and C4). In vivo experiments have been successfully performed with 4 h and 12 h of labeling, and in vitro experiments have been performed with 2-12 h of labeling.
  4. In mice experiments, doxycycline can also be administered through drinking water (Step C3).
  5. This protocol has been successfully used in mouse lungs, brain and mammary fat pad. Other organs have not been tested yet. 
  6. For in vivo experiments, the growth curve of different cell lines can vary. Hence, it is important to pick the time for 5-FC injection based on desired size of the tumors. For example, mouse cancer cell lines grow much faster than MDA-MB-231 cells, so earlier time point might be suitable for mouse cancer cell lines (Step C4).
  7. The strain, sex and age of the mice can vary for different cell lines. For example, different mice strain might be suitable for syngeneic experiments.

Recipes

  1. Cell lysis buffer
    20 mM Tris-HCl pH 7.5
    500 mM LiCl
    1% LiDS
    1 mM EDTA
    5 mM DTT (DTT is added freshly)
  2. mRNA Wash Buffer I
    20 mM Tris-HCl pH 7.5
    500 mM LiCl
    0.1% LiDS
    1 mM EDTA
    5 mM DTT (DTT is added freshly)
  3. mRNA Wash Buffer II
    20 mM Tris-HCl pH 7.5
    500 mM LiCl
    1 mM EDTA
  4. mRNA Wash Buffer III
    20 mM Tris-HCl pH 7.5
    200 mM LiCl
    1 mM EDTA
  5. Elution Buffer
    20 mM Tris-HCl pH 7.5
    1 mM EDTA
  6. SSPET buffer
    1x SSPE (Sodium Chloride-Sodium Phosphate-EDTA)
    0.05% Tween 20
  7. TE buffer
    10 mM Tris pH 7.5
    1 mM EDTA

Acknowledgments

H.B. was supported by a Damon Runyon Postdoctoral Fellowship. This work was supported by NIH grants P01-CA094060 (J.M.), P30-CA008748 (MSKCC), and a DOD Innovator award W81XWH-12-0074 (J.M.). The protocol is derived from Basnet et al., 2019.

Competing interests

H.B and J.M have filed for patent for Flura-seq method (PCT/US18/22092). J.M serves in the scientific advisory board and owns company stock in Scholar Rock.

Ethics

Mouse experiments were performed following the protocol (99-09-032) approved by the MSKCC Institutional Animal Care and Use Committee (IACUC) that is valid until 06/19/2020.

References

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  2. Bertin, B., Renaud, Y., Aradhya, R., Jagla, K. and Junion, G. (2015). TRAP-rc, translating ribosome affinity purification from rare cell populations of Drosophila embryos. J Vis Exp(103).
  3. Cleary, M. D., Meiering, C. D., Jan, E., Guymon, R. and Boothroyd, J. C. (2005). Biosynthetic labeling of RNA with uracil phosphoribosyltransferase allows cell-specific microarray analysis of mRNA synthesis and decay. Nat Biotechnol 23(2): 232-237.
  4. Gay, L., Karfilis, K. V., Miller, M. R., Doe, C. Q. and Stankunas, K. (2014). Applying thiouracil tagging to mouse transcriptome analysis. Nat Protoc 9(2): 410-420.
  5. Gay, L., Miller, M. R., Ventura, P. B., Devasthali, V., Vue, Z., Thompson, H. L., Temple, S., Zong, H., Cleary, M. D., Stankunas, K. and Doe, C. Q. (2013). Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA. Genes Dev 27(1): 98-115.
  6. Heiman, M., Schaefer, A., Gong, S., Peterson, J. D., Day, M., Ramsey, K. E., Suárez-Fariñas, M., Schwarz, C., Stephan, D. A., Surmeier, D. J., Greengard, P. and Heintz, N. (2008). A translational profiling approach for the molecular characterization of CNS cell types. Cell 135(4): 738-748.
  7. Hida, N., Aboukilila, M. Y., Burow, D. A., Paul, R., Greenberg, M. M., Fazio, M., Beasley, S., Spitale, R. C. and Cleary, M. D. (2017). EC-tagging allows cell type-specific RNA analysis. Nucleic Acids Res 45(15): e138.
  8. Machado, L., Esteves de Lima, J., Fabre, O., Proux, C., Legendre, R., Szegedi, A., Varet, H., Ingerslev, L. R., Barres, R., Relaix, F. and Mourikis, P. (2017). In situ fixation redefines quiescence and early activation of skeletal muscle stem cells. Cell Rep 21(7): 1982-1993.
  9. Matsushima, W., Herzog, V. A., Neumann, T., Gapp, K., Zuber, J., Ameres, S. L. and Miska, E. A. (2018). SLAM-ITseq: sequencing cell type-specific transcriptomes without cell sorting. Development 145(13). pii: dev164640.
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  11. Mullen, C. A., Kilstrup, M., and Blaese, R. M. (1992). Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system. Proc Natl Acad Sci U S A 89(1): 33-37.
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简介

组织由不同的细胞类型组成,它们的相互作用会引发不同的基因表达模式,从而调节组织的形成,再生,体内平衡和修复。这些基因表达模式的分析需要能够在其组织微环境中尽可能接近地捕获目标细胞的转录组的方法。设计用于原位转录组学的当前技术受到其低灵敏度的限制,该技术要求细胞类型占总组织的1%以上,因此很难对从其天然细胞中快速分离的稀有细胞群体进行转录分析微环境。为了解决这个问题,我们开发了利用尿嘧啶脱氨酶(CD)将非天然嘧啶氟胞嘧啶转化为氟尿嘧啶的氟尿嘧啶标记的RNA测序(Flura-seq)。 S的表达。啤酒酵母CD和暴露于氟胞嘧啶会产生氟尿嘧啶,并代谢标记特定目的细胞中新合成的RNA。然后可以对含氟尿嘧啶标记的RNA进行免疫纯化,并用于下游分析。在这里,我们描述了在体外 和体内 执行Flura-seq的详细协议。 Flura-seq的坚固性,简单性和无毒性使该工具广泛适用于发育,再生和癌症生物学的许多研究。
(背景)在器官中,细胞与组织微环境相互作用,其中包括器官特异性的驻留细胞,免疫细胞,血管周围壁ni,细胞外基质,细胞因子,代谢产物和氧浓度范围。这些相互作用决定了通常在细胞中具有功能意义的特定基因组的表达。与这些相互作用相关的转录反应通常是动态的,只有在相互作用得以保留的情况下才能观察到。因此,维持组织微环境以确保在生理条件下捕获细胞的真实转录状态至关重要。

目前研究细胞类型特异性转录组的技术存在局限性,使其无法有效地用于研究稀有和代表性不足的细胞群体。单细胞RNA测序(scRNA-seq)进行或不进行荧光激活细胞分选(FACS)步骤,都需要对组织进行大量的物理和酶处理,这不仅破坏了宿主微环境对细胞的影响,而且还施加了压力在这些细胞上,其严重破坏了从分离的细胞的转录组中辨别宿主基质的影响的能力。据报道,在肌肉干细胞的FACS加工过程中,多达7500个基因发生了两倍以上的改变,用于转录组学分析(Machado et al。,2017)。 原位转录组图谱分析消除了这些问题,但对代表不到1%组织的细胞群缺乏必要的敏感性。例如,翻译核糖体亲和纯化和mRNA测序(TRAP-Seq)(Heiman等人,2008年)不适合分析构成总群体不到1%的细胞(白蛋白> et al。,2015; Obenauf et al。,2015)。在感兴趣的细胞中,直接用基于硫基尿嘧啶(TU)和乙炔基胞嘧啶(EC)的RNA进行基于酶的直接代谢标记,由于缺乏对这些酶的细胞进行RNA附带标记,因此敏感性和特异性受到限制,并且需要在体外进行 < / em>生物素化步骤(Cleary等,2005年; Miller等,2009年; Gay等,2013年和2014年; Hida et al。,2017)。 TU标签的敏感度限制为5%(Gay et al。,2013)。硫醇(SH)链接的组织中RNA的代谢标记烷基化(SLAM-ITseq)消除了与TU标记方法中未标记硫醇的RNA纯化相关的噪音(Matsushima 等人。,2018),但是在缺乏UPRT表达的细胞中通过内源酶进行不希望的TU标签仍然是一个限制。我们描述了Flura-seq的发展,这是一种基于胞嘧啶脱氨酶(CD)的稀有细胞群体原位转录组谱分析方法,仅占器官的0.003%(Basnet et等,2019)。 Flura-seq要求CD的外源表达,CD是真菌和原核生物中嘧啶挽救途径的关键酶(Mullen等人,1992年),而em中则是UPRT。刚地。 CD在哺乳动物细胞中不存在,它们出于相同的目的而使用胞苷脱氨酶(Mullen等人,1992)。除了将胞嘧啶转化为尿嘧啶外,CD还可以将非天然嘧啶5-氟胞嘧啶(5-FC)转化为5-氟尿嘧啶(5-FU)。 5-FU内源性转化为三磷酸氟尿苷(F-UTP),然后将其掺入RNA。 UPRT的共表达限制了表达CD的细胞中RNA的标记(Basnet et al。,2019)。 Flura-seq可用于体外共培养实验以及体内实验,其中完整组织中存在表达CD-UPRT的目标细胞。 Flura-seq技术上非常简单,只需要非常基本的分子生物学技能,并且整个过程可以在24小时内完成。在这里,我们描述了用于体外和体内实验的Flura-seq的RNA标记和分离的详细协议。

关键字:原位转录组学, 胞嘧啶脱氨酶, 尿嘧啶磷酸核糖转移酶, 氟尿嘧啶, 新生RNA

材料和试剂

  1. 5 ml聚苯乙烯圆底管(Corning,目录号:352058)
  2. 60 mm组织培养板(Corning,目录号:353004)
  3. Oligo(dT) 25 磁珠(New England Biolabs,目录号:S1419S)
  4. Protein G Dynabeads(Thermo Fisher Scientific,目录号:10004D)
  5. 6-8周大的无胸腺裸雌鼠(Envigo,货号:069)
  6. 强力霉素饮食(625 mg / kg,Envigo,目录号:TD.07383)
  7. BrdU抗体(Abcam,目录号:ab6326)
  8. CD-UPRT(Addgene,质粒编号:126677)
  9. rtTA3(Addgene,质粒编号:26730)
  10. 强力霉素(Sigma-Aldrich,目录号:D9891)
  11. 5-氟胞嘧啶(5-FC)(Sigma-Aldrich,目录号:F7129)
  12. 5-Bromo-2﹑-脱氧尿苷(BrdU)(Millipore Sigma,目录号:B5002)
  13. 胸腺嘧啶(Sigma-Aldrich,目录号:T0376)
  14. RNeasy MinElute清洁套件(Qiagen,目录号:74204)
  15. cDNA试剂盒-First Strand Transcriptor(Roche,目录号:043790-12001)
  16. RNeasy Mini Kit(Qiagen,目录号:74106)
  17. 乙二胺四乙酸溶液(EDTA)(Sigma-Aldrich,目录号:03690)
  18. 吐温20(Sigma Millipore,目录号:P1379)
  19. 十二烷基硫酸锂(LiDS)(Sigma Millipore,目录号:L4632)
  20. 氯化锂(LiCl)(Sigma Millipore,目录号:203637)
  21. 超纯BSA(BSA)(Thermo Fisher Scientific,目录号:AM2616)
  22. 糖原(Thermo Fisher Scientific,目录号:R0551)
  23. RNAlater(Sigma-Aldrich,目录号:R0901)
  24. 20x UltraPure SSPE缓冲液(Thermo Fisher Scientific,目录号:15591043)
  25. PBS
  26. Tris基地
  27. 盐酸
  28. 乙醇(Decon Labs,目录号:2716)
  29. DTT(Sigma-Aldrich,目录号3483-12-3)
  30. 细胞裂解缓冲液(请参阅食谱)
  31. mRNA洗涤缓冲液I(请参阅食谱)
  32. mRNA Wash Buffer II(请参阅食谱)
  33. mRNA Wash Buffer III(请参阅食谱)
  34. 洗脱缓冲液(请参阅配方)
  35. SSPET缓冲液(请参阅配方)
  36. TE缓冲区(请参阅食谱)

设备

  1. PRO Scientific Inc(均质器)的PRO 200研磨机(PRO Scientific,目录号:01-01200)
  2. 磁力架(ThermoFisher Scientific,目录号:12321D)
  3. Thermo Scientific旋转器(ThermoFisher Scientific,目录号:88881001)
  4. Eppendorf Thermomixer(ThermoFisher Scientific,目录号:05-400-200)
  5. 离心机

程序

  1. 使CD-UPRT表达稳定细胞
    1. 通过使用标准协议用CD-UPRT(Addgene)和rtTA3(Addgene)慢病毒转导细胞来生成感兴趣的表达CD-UPRT的细胞系(例如MDA-MB-231)。

  2. 体外
    的RNA标记 第一天
    1. 在3mm体积的60 mm平板中,将表达多西环素诱导性CD-UPRT的1,000个细胞(例如MDA-MB-231)与100万个4T1细胞(或其他细胞(可选))一起以3毫升的体积接种(强力霉素诱导系统为可选的)。
    第二天
    1. 加入1微克/毫升强力霉素。
    第3天
    1. 强力霉素处理后24小时,分别将5-FC和胸腺嘧啶添加至250μM(50 mM储备液)和125μM(25 mM储备液)终浓度。 5-FC和胸腺嘧啶原液是在PBS中制得的。
    2. 2-12小时后,使用细胞裂解缓冲液收获细胞。用3 ml PBS洗涤细胞1x,然后在平板上添加1 ml细胞裂解缓冲液。在1.5 ml eppendorf管中刮擦细胞。

  3. 小鼠中RNA的标记
    第一天
    1. 在PBS中制备500,000 / ml表达CD-UPRT的稳定细胞(上文A.1中所述)。
    2. 通过尾静脉向小鼠注射100μl(50,000个细胞)。
    第28天
    1. 如果细胞表达强力霉素诱导的CD-UPRT,则在5-FC注射前3天将小鼠饮食改为强力霉素饮食。如果细胞组成性表达CD-UPRT,则跳过此步骤。
    第31天
    1. 腹腔内给小鼠注射250μg/ kg 5-FC的PBS(12.5μg/ ml),皮下注射125μg/ kg胸腺嘧啶的PBS(6.25 g / ml)。
    2. 4-12小时后,通过CO 2 窒息对小鼠实施安乐死,收获器官,并将器官保留在RNAlater缓冲液中。
    3. 将感兴趣的器官(例如,肺)放入5 ml聚苯乙烯圆底管中的2 ml细胞裂解缓冲液(配方1)中,然后用组织研磨机将器官均质化。
    4. 将裂解液平均分为两个试管,并在每个试管中加入3 ml细胞裂解缓冲液(裂解液可在液氮中速冻以备将来使用)。

  4. &nbsp; mRNA的分离
    1. 在C.7的裂解物中添加1x体积的细胞裂解缓冲液(4 ml),在室温(RT)旋转5分钟。对于体外细胞,添加适当体积的裂解缓冲液(对于60 mm融合板,为1 ml),并刮下细胞。
    2. 将裂解液放入1.5 ml Eppendorf管中,在室温下以16,000 x g 离心5分钟,并保留上清液。
    3. 准备oligo dT 25 珠(﹄100μl珠/〜25 mg肺/脑)。
      1. 将250μl珠子/ Eppendorf管(1.5 ml)放在磁力架中。
      2. 卸下存储缓冲区。
      3. 上下吹打五次,用1 ml细胞裂解缓冲液洗涤小珠1x。
    4. 从D.2中取出上清液(1 ml / 250μl珠子),并将其添加到新鲜制备的oligo dT 25 珠子中。
    5. 在旋转仪中于室温孵育10分钟。
    6. 丢弃上清液。
    7. 通过在室温下上下吸移7次,用1 ml mRNA洗涤缓冲液I(配方2)洗涤珠子2x。
    8. 在室温下,通过上下吹打七次,用1 ml mRNA洗涤缓冲液II(配方3)洗涤小珠2x。
    9. 在室温下,通过上下吹打七次,用1 ml mRNA洗涤缓冲液III(配方4)将珠子洗涤1x。
    10. 在室温下通过上下吹打七次,用1 ml TE缓冲液(配方7)将珠子洗1次。
    11. 加入50μl洗脱缓冲液(配方5),并在Eppendorf Thermomixer中于85°C洗脱RNA,以750 rpm摇动2分钟。
    12. 将珠子放在磁性架中,并保留洗脱液。 mRNA可以储存在-80 80C以便将来使用。

  5. 5-FU标记的mRNA的纯化
    1. 在室温下每个样品准备70μl蛋白G珠。将珠子放在磁力架中,然后取出存储缓冲液。最多可将350μl(用于5个样品)的珠子转移到一个1.5 ml的Eppendorf管中。
    2. 在室温下,通过上下吹打五次,用1 ml的0.5x SSPET缓冲液(配方6)洗涤磁珠。
    3. 用1 ml 0.5x SSPET缓冲液(含10μg/ ml BSA和20μg/ ml糖原)在4°C下封闭小珠1小时。
    4. 通过在室温下上下吹打五次,用1 ml的0.5x SSPET缓冲液洗涤珠子1x。
    5. 在1.5毫升Eppendorf管中的750升0.5x含BrdU抗体的SSPET缓冲液(1克用于RT-PCR,5克用于RNA-seq)中添加35珠。在4°C下旋转过夜。
    6. 在室温下通过上下吹打五次,用1 ml的0.5x SSPET缓冲液洗涤珠子1x。
    7. 加入来自步骤D12的35微升珠子的mRNA(在这种情况下为400微升),用相同体积(400微升)的1x SSPET缓冲液稀释。总体积约为835升。
    8. 在室温下使用ThermoScientific旋转器旋转2小时。
    9. 通过在室温下上下吹打7次,用1 ml的0.5x SSPET缓冲液洗涤2x。
    10. 通过在室温下上下吹打7次,用1 ml 1x SSPET缓冲液洗涤2x。
    11. 在室温下通过上下吹打七次,用1 ml TE缓冲液洗涤1次。
    12. 在室温下,在Eppendorf Thermomixer中的TE缓冲液中以200μl100μg/ ml BrdU洗脱结合的mRNA,在900 rpm的温度下洗脱45分钟。
    13. 保留上清液(5-FU标记的mRNA)。
    14. 将200升上清液与RNeasy试剂盒中的700升RLT缓冲液混合。
    15. 加入500μl100%的乙醇,并充分混合。
    16. 将溶液通过RNeasy色谱柱。
    17. 用RNeasy试剂盒中的500μlRPE缓冲液洗涤1次。
    18. 用500μl80%乙醇洗涤1次。
    19. 在室温下以13,000 x g 旋转2分钟。
    20. 用200μl不含RNase的水洗脱RNA。
    21. 加入600升0.5x SSPET洗脱液。
    22. 如步骤E6所述,洗涤与BrdU抗体一起孵育的Protein G Dynabeads。
    23. 如步骤E7-E13中所述,重新免疫沉淀5-FU标记的mRNA。
    24. 如步骤E14-E19所述使用RNeasy MiniElute Cleanup试剂盒纯化RNA,并用12.5μl不含RNase的水洗脱RNA。
    25. 进行cDNA合成以进行RT-PCR或RNA测序。

  6. 共移植细胞和小鼠肺中5-氟尿嘧啶(5-FU)标记的mRNA的标记和纯化
    将CD-UPRT(Addgene)和rtTA3(Addgene)(MDA231-CDUPRT)转导的1,000个人MDA-MB-231细胞与100万小鼠4T1细胞共培养。如B节所述标记RNA,并如D节和E节所述纯化5-FU标签的mRNA。具有代表性的人和小鼠基因的5-FU标签mRNA相对于非免疫纯化的mRNA的富集度通过RT- PCR(图1A)。
    &nbsp;通过尾静脉将5 x 10 4 MDA231-CDUPRT细胞注入小鼠肺。 28天后,用强力霉素饮食喂养小鼠3天,诱导CD UPRT表达,如C节所述标记RNA,如D和E节所述纯化5-FU标记的mRNA。5-FU富集通过RT-PCR测量相对于非免疫纯化的mRNA的代表性人和小鼠基因的带标签的mRNA(图1B)。


    图1.通过胞嘧啶脱氨酶表达标记和分离5-FU标记的RNA。将1,000个表达CD-UPRT的人MDA-MB-231细胞与100万小鼠4T1细胞共培养,按照方案中所述的方法从人细胞中分离出5-FU标记的mRNA,并显示了相对于其相应的2%输入(未免疫纯化的mRNA)的代表性人基因和小鼠基因的富集(n = 3,SEM)。 B.通过尾静脉将表达CD-UPRT的50,000个MDA-MB-231细胞通过尾静脉注射到肺中,并在28天后按照方案中所述的方法分离了5-FU标记的mRNA,并富集了代表性的人和小鼠基因通过RT-PCR(n = 6,(SEM)测量了1%的输入(非免疫纯化的mRNA)。 ﹒h‧表示人类,﹒m‧表示鼠标。

笔记

  1. 对于体外实验,细胞融合不会影响5-FU标签(步骤B1),该方法可能适用于所有细胞类型(贴壁细胞和悬浮细胞)。我们已经将该方法成功应用于MDA-MB-231,HCC1954,H2087、293T和4T1细胞。
  2. 胸腺嘧啶不能完全溶解在PBS中。使用前过滤溶液(步骤B3)。
  3. 5-FC和胸腺嘧啶的孵育时间更长,可增加RNA标记,并产生更高的信噪比。对于非常少量的细胞(小于0.01%或小于1,000个细胞),建议标记12小时。对于超过1 x 10 5 细胞组成的条件,RNA标记4 h就足够了。 (步骤B4和C4)。分别在4小时和12小时的标记下进行了体内实验,而在2-12小时的标记下进行了体外实验。
  4. 在小鼠实验中,强力霉素也可以通过饮用水给药(步骤C3)。
  5. 该协议已成功用于小鼠肺部,大脑和乳腺脂肪垫。其他器官尚未经过测试。
  6. 对于体内实验,不同细胞系的生长曲线可能会有所不同。因此,重要的是要根据所需的肿瘤大小来选择5-FC注射时间。例如,小鼠癌细胞系的生长要比MDA-MB-231细胞快得多,因此更早的时间点可能适用于小鼠癌细胞系(步骤C4)。
  7. 小鼠的品系,性别和年龄可能因不同的细胞系而异。例如,不同的小鼠品系可能适合进行同系实验。

菜谱

  1. 细胞裂解缓冲液
    20 mM Tris-HCl pH 7.5
    500 mM氯化锂
    1%LiDS
    1毫米EDTA
    5 mM DTT(新鲜添加DTT)
  2. mRNA洗涤缓冲液I
    20 mM Tris-HCl pH 7.5
    500 mM氯化锂
    0.1%LiDS
    1毫米EDTA
    5 mM DTT(新鲜添加DTT)
  3. mRNA Wash Buffer II
    20 mM Tris-HCl pH 7.5
    500 mM氯化锂
    1毫米EDTA
  4. mRNA Wash Buffer III
    20 mM Tris-HCl pH 7.5
    200 mM氯化锂
    1毫米EDTA
  5. 洗脱缓冲液
    20 mM Tris-HCl pH 7.5
    1毫米EDTA
  6. SSPET缓冲液
    1x SSPE(氯化钠-磷酸钠-EDTA)
    0.05%吐温20
  7. TE缓冲液
    10 mM Tris pH 7.5
    1毫米EDTA

致谢

H.B.获得了Damon Runyon博士后奖学金的支持。 NIH授予P01-CA094060(J.M。),P30-CA008748(MSKCC)和国防部创新奖W81XWH-12-0074(J.M.)来支持这项工作。该协议源自Basnet et al。,2019年。

利益争夺

H.B和J.M已申请Flura-seq方法的专利(PCT / US18 / 22092)。 J.M在科学顾问委员会任职,并拥有Scholar Rock公司股票。

伦理

遵循MSKCC机构动物护理和使用委员会(IACUC)批准的有效期至06/19/2020的协议(99-09-032)进行小鼠实验。

参考文献

  1. Basnet,H.,Tian,L.,Ganesh,K.,Huang,Y.H.,Macalinao,D.G.,Brogi,E.,Finley,L.W.和Massagu用,J.(2019)。 Flura-seq可以识别早期转移性定植过程中器官特异性的代谢适应。 Elife 8:e43627。
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Copyright Basnet and Massague. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Basnet, H. and Massague, J. (2019). Labeling and Isolation of Fluorouracil Tagged RNA by Cytosine Deaminase Expression. Bio-protocol 9(22): e3433. DOI: 10.21769/BioProtoc.3433.
  2. Basnet, H., Tian, L., Ganesh, K., Huang, Y. H., Macalinao, D. G., Brogi, E., Finley, L. W. and Massagué, J. (2019). Flura-seq identifies organ-specific metabolic adaptations during early metastatic colonization. Elife 8: e43627.
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