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May 2017

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Comprehensive Identification of Translatable Circular RNAs Using Polysome Profiling
利用多聚体谱综合鉴定可翻译环状RNA   

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Abstract

Circular RNAs (circRNAs), a special type of RNAs without 5’- and 3’-ends, are widely present in eukaryotes and known to function as noncoding RNAs to regulate gene expression, including as miRNA sponges. Recent studies showed that many exonic circRNAs, generated by back-splicing of pre-mRNAs, can be translated in a cap-independent fashion through IRESs or m6A RNA methylation. However, the scope of the translatable circRNAs and the biological function of their translation products are still unclear in different cells and tissues. Ribosome footprinting and proteomic analysis were usually used to globally identify translatable circRNAs. However, both methods have low sensitivity due to the low efficiency in the discovery of circRNA specific reads or peptides (i.e., the back-splicing junctions are difficult to recover by the short reads of ribosome footprinting and the limitation of proteomic analysis). Here, we described an alternative method to identify translatable circRNAs using polysome profiling and circRNA-seq. Generally, polysome-associated RNAs were separated with sucrose gradients. Then polysome-bound circRNAs were enriched by an RNase R treatment and identified through paired-end deep sequencing. Thus, this method is more sensitive than ribosome footprint and proteomic analyses for the identification of translatable circRNAs.

Keywords: Polysome profiling (多核糖体剖析), circRNA translation (circRNA 翻译), RNA-seq (RNA-seq), Back-splicing junction (反向剪接点), RNase R (RNase R)

Background

Circular RNAs (circRNAs) are a special type of RNA without 5’- and 3’-ends, which form a covalent close loop. They were first discovered in viroids four decades ago (Sanger et al., 1976). Recently, many studies showed that circRNAs were widely present in eukaryotes and expressed in a tissue-specific manner (Rybak-Wolf et al., 2015; Chen, 2020). Several types of circRNAs are found in cells, including exonic circRNA, EIciRNA, and ciRNA (Kristensen et al., 2019); however, the majority of them are exonic circRNAs that are generated through back-splicing of pre-mRNAs (Salzman et al., 2012 and 2013; Memczak et al., 2013; Ashwal-Fluss et al., 2014; Zhang, X. O. et al., 2014). Thus, most exonic circRNAs sequences are the same as those of linear mRNAs, and the back-splicing junctions are unique sequences for circRNA detection.


CircRNAs regulate biological processes through their activities as miRNA sponges, protein scaffolds, and transcription regulators (Kristensen et al., 2019). However, the functions of most circRNAs are still unclear. Recently, several studies showed that many circRNAs can be translated through cap-independent translation from IRESs or m6A RNA modifications (Legnini et al., 2017; Pamudurti et al., 2017; Yang et al., 2017). In addition, some circRNA encode peptides reported to influence cancer proliferation or lifespan in flies (Weigelt et al., 2020; Yang et al., 2018; Zhang, M. et al., 2018), indicating that the translation products from circRNAs play important biological roles. However, the roles of translatable circRNAs are still unknown. Therefore, transcriptome-wide identification of translatable circRNAs is an important way to study their biological function.


Two different types of omics approaches are usually used to comprehensively measure the translation of mRNAs at RNA (translatome) and protein (proteome) levels. Ribosome footprinting is an emerging technique to globally measure mRNA translation via high-throughput sequencing of ribosome-protected mRNA fragments; however, it’s difficult to capture the translating circRNAs. Since the ribosome-protected mRNA fragments are short (around 28-35nt), the back-splicing junctions are easy to be missed by this method. Proteomic analysis is a technique to directly determine the translation products of mRNAs through identification of enzymatically digested peptides. Similar to ribosome footprinting, the peptides coded by back-splicing junctions are difficult to identify through tandem mass spectrometry.


We developed a new method to identify translatable circRNAs using polysome profiling and circRNA-seq. The polysome-associated RNAs can be separated using sucrose gradients and then isolated from different fractions. The polysome-bound circRNAs are enriched by an RNase R treatment and used to generate the circRNA-seq libraries. The resulting libraries are pair-end sequenced, and the circRNAs are identified by the back-splicing junction reads. Therefore, our method is easier and more sensitive than ribosome footprint and proteomic analyses for identifying translatable circRNAs.

Materials and Reagents

  1. 10 cm and 15 cm dish (Thermo ScientificTM, NuncTM EasYDish, catalog numbers: 150464, 150468)

  2. 50 ml and 15 ml centrifuge tube (Thermo ScientificTM, Thermo ScientificTM NuncTM, catalog numbers: 339650, 339652)

  3. 1.5 ml and 2 ml Eppendorf tube (RNase free) (CORNING, Axygen, catalog numbers: MCT-150-C, MCT-200-C)

  4. 10 ml and 25 ml serological pipette (Thermo ScientificTM, NuncTM, catalog numbers: 170357N, 170356N)

  5. 10 ml syringe (Sigma-Aldrich, catalog number: Z248029)

  6. Pipetting needle (BioComp, belong to Gradient Master in Equipment)

  7. 25 cm cell scraper (JET BIOFIL, catalog number: CSC011025)

  8. Polypropylene centrifuge tube (Beckman coulter, catalog number: 331372)

  9. Vacuum Filtration Systems (Corning, catalog number: 430758)

  10. Cell line: HEK293T

  11. Nuclease free water (Sigma-Aldrich, catalog number: W4502)

  12. TRIzolTM reagent (Thermo Fisher, catalog number: 15596018)

  13. Chloroform (Sinoreagent, catalog number: 10006818)

  14. Isopropanol (Sinoreagent, catalog number: 80109218)

  15. Ethanol (Sigma-Aldrich, catalog number: E7023)

  16. Dulbecco’s modified eagle medium (DMEM) (meilunbio, catalog number: MA0212)

  17. Phosphate buffered saline (PBS) (Hyclone, catalog number: SH30256.01)

  18. Ribonuclease inhibitor (Promega, catalog number: N2518)

  19. RQ1 RNase-free DNase (Promega, catalog number: M6101)

  20. RNase R (Lucigen, catalog number: RNR07250)

  21. cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Roche, catalog number: 11873580001)

  22. Cycloheximide (CHX) (Sigma, catalog number: C7698)

  23. Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: 43815)

  24. TritonTM X-100 (Sigma-Aldrich, catalog number: T8787)

  25. Bromophenol blue (Sigma-Aldrich, catalog number: B0126)

  26. KAPA Stranded RNA-Seq Kit with RiboErase (HMR) (KAPA, catalog number: KK8483)

  27. RNA clean and concentrator kit (ZYMO RESEARCH, catalog number: R1019)

  28. 10× Polysome buffer (see Recipes)

  29. Lysis buffer (see Recipes)

  30. Wash buffer (see Recipes)

  31. 70% sucrose solution (see Recipes)

  32. 50% sucrose grading solution (see Recipes)

  33. 10% sucrose grading solution (see Recipes)

Equipment

  1. Ultracentrifuge (with rotor SW41) (Beckman, model: Optima XPN-100-IVD, catalog number: A99846)

  2. Photo-spectrometer (Thermo Fisher, NanoDrop, catalog number: ND-1000)

  3. Density gradient fractionation system (Complete system includes tube piercing system; syringe pump; UA-6 Detector with 254 and 280 nm filters, R1 Fraction Collector, Density Gradient Flow Cell, cables, and tubing) (BRANDEL, catalog number: BR-188)

  4. Gradient Master (BioComp, catalog number: Biocomp 108)

Software

  1. Peak chart (Data capture software for Density gradient fractionation system, www.brandel.com)

  2. TopHat 2/ TopHat-Fusion (https://ccb.jhu.edu/software/tophat/index.shtml, https://ccb.jhu.edu/software/tophat/fusion_index.shtml)

  3. CIRCexplorer2 (https://circexplorer2.readthedocs.io/en/latest/)

Procedure

  1. Preparation of sucrose gradient (Figure 1)

    1. Prepare 500 ml of 10× polysome buffer in nuclease free water and 500 ml of 70% (w/v) sucrose solution in 1× polysome buffer (see Recipes). Filter solutions with Vacuum Filtration Systems.

    2. Prepare 10% (w/v) and 50% (w/v) sucrose solution with 70% (w/v) sucrose solution (see Recipes).

    3. Add the 10% and 50% sucrose solution, in that order, into a Beckman polypropylene centrifuge tube at room temperature.

      Note: Add a half tube of 10% sucrose solution first and then the 50% sucrose solution to the bottom of the tube under the 10% solution using a needle.

    4. Load the centrifuge tube on the gradient master to make a 10-50% (w/v) sucrose gradient. Sucrose gradients can be used immediately or stored long-term at -80°C (no more than 2 weeks) (see Note 1).



      Figure 1. Protocol for Sucrose Gradient Preparation


  2. Sample preparation

    Note: Cool all the solutions and buffers before use. Keep the samples on ice all the time if possible.

    1. Seed the cells in 10 cm or 15 cm dishes one day before the experiment (see Note 2 and Table 1).


      Table 1. Recommend schedule of this protocol



    2. Add 100 μg/ml cycloheximide into the culture medium and incubate cells at 37°C for 10 min.

    3. Discard the medium and wash the cells with 10 ml ice-cold PBS containing 100 µg/ml cycloheximide twice.

    4. Add 10 ml ice-cold PBS to cells and harvest the cells with cell scraper.

    5. Transfer the cell suspension into 15 ml centrifuge tube and centrifuge at 1,000 × g for 3 min at 4°C.

    6. Remove the supernatant and add 250 μl lysis buffer to 15 ml centrifuge tube.

    7. Lyse the cells by gently pipetting and transfer the lysate into a 1.5 ml Eppendorf tube.

    8. Keep cell lysate on ice for 10 min and centrifuge the lysate at 14,000 × g for 10 min at 4°C to remove cell debris.

    9. Transfer the supernatant into a new 1.5 ml Eppendorf tube.

    10. Dilute 1 μl samples with 999 μl water, and measure the approximate RNA concentration using a NanoDrop photo-spectrometer (see Note 3).

    11. Keep the samples on ice for immediate use or freeze them in liquid nitrogen immediately and store at -80°C until use (no more than one month).


  3. Polysome profiling

    1. Remove the same volume of solution as that of the loading sample from the top of the 10-50% sucrose gradient and then load the samples onto the sucrose gradient. The optimal loading volume is 200-500 μl (see Note 3). Keep 10% of lysates as an input.

    2. Centrifuge at 35,000 × g for 2.5h at 4°C using the SW41 rotor in a Beckman Ultracentrifuge.

    3. Set up the density gradient fractionation system while the samples are centrifuging. Fill the syringe with chasing solution [70% (w/v) sucrose with bromophenol blue], flush the line, and calibrate the ultraviolet spectrophotometer following the equipment instructions.

    4. Gently remove tubes from the rotor and put them into the density gradient fractionation system. Alternatively, keep them at 4°C until use (no more than 4 h).

    5. Load the sample under the UA-6 Detector and puncture the bottom of the tube, communicating the syringe pump and detector system (see Figure 2).



      Figure 2. Protocol for Density Gradient Fractionation System Set-off


    6. Number the 2 ml Eppendorf tubes and place them onto the R1 Fraction Collector.

      Note: About 750 μl of sample will be collected in each tube.

    7. Set syringe pump at 1.5 ml/min flow rate and switch on remote model. Set the fraction collector by time.

    8. Turn on density gradient fractionation system using Peak chart software. Record the data with Peak chart (see Figure 3) and collect the fractions with the fraction collector.

    9. Store the samples at -80°C until use (no more than one month).



      Figure 3.Example of Polysome Profiling. The absorbance of OD254 (A254) that represents the abundance of RNA fragments was measured by UA-6 detector. The sucrose gradient was fractionated into 17 fractions with Density gradient fractionation system. The value of A254 was recorded by Peak Chart and plotted as shown.


  4. RNA-seq library preparation

    1. Add 750 μl of TRIzol into each tube, and extract the RNAs according to the manufacturer’s instruction (use chloroform, isopropanol, and 70% ethanol to separate RNAs from samples and elute in about 30 μl water) or store at -80°C.

    2. Pool RNA samples in free, monosome, light polysome or heavy polysome fractions with equal volume, respectively (e.g., as shown in figure 3, fraction 1 was termed “free fraction,” fraction 6-7 was termed “monosome fraction,” fractions 8-10 were pooled with equal volumes and termed “light polysome fraction,” and fractions 11-16 were pooled with equal volume and termed “heavy polysome fraction”).

    3. Measure the RNA concentration of each RNA mixture with NanoDrop.

    4. Treat the RNAs with DNase I (RNase free) at 37°C for 15 min.

      2 μg of RNA                     x μl

      DNase I                             1μl

      10× DNase I buffer         5 μl

      Water                          44-x μl

    5. Clean up the RNAs using the ZYMO RNA clean and concentrator kit according to the manufacturer’s instructions.

    6. Treat the DNase I treated RNAs with RNase R at 37°C for 30 min (see Note 4).

      1 μg of RNA                          x μl

      RNase R                               1 μl

      10× RNase R buffer           5 μl

      Water                              44-x μl

    7. Clean up the RNAs using the ZYMO RNA clean and concentrator kit according to the manufacturer’s instruction (eluted RNAs in 20 μl water).

    8. Construct the RNA-seq libraries with the KAPA stranded RNA-seq kit with RiboErase according to the manufacturer’s instruction.

    9. Sequence the libraries using a 150 bp paired-end approach with a 100 million read depth per sample.

      Note: We recommend Illumina HiSeq® 3000/HiSeq 4000 Sequencing Systems for RNA-seq.


  5. Identification of translatable circRNAs

    1. Perform quality control on raw sequencing reads and trim the adaptor sequences. Discard the low-quality reads. Trim the adaptor sequences from the reads and map them to the genome.

    2. Align the reads to the reference genome with TopHat2, and map the unmapped reads with TopHat-Fusion.

    3. Annotate and assembly the circRNA transcripts using the CIRCexplorer2 pipeline (see details in GitHub: https://circexplorer2.readthedocs.io/en/latest/) (Zhang, X. O. et al., 2016).

Notes

  1. If there is not Gradient Master for the gradient forming, we suggest an alternative method to make gradient through freezing and thawing. To make a 10-50% (w/v) sucrose gradient, prepare 50% (w/v), 40% (w/v), 30% (w/v), 20% (w/v), and 10% (w/v) sucrose solutions. Put 2 ml of 50% sucrose into a Beckman centrifuge tube and freeze on dry ice-ethanol, then put the 40% solution (2 ml) and also freeze, and repeat with the other solutions. The tubes can be frozen and stored at -80°C. One day before use, thaw the tubes in the refrigerator (4°C) overnight, and they are ready to use.

  2. To keep the cells with active translation, it is better to harvest them at around 70-80% confluency. For example, for HEK293T, cells can be seeded in 10 cm (2.2 × 106 cells) or 15 cm (5.0 × 106 cells) dishes one day before the experiment and harvested at about 7.0 × 106 cells/10 cm dish or 16.0 × 106 cells/ 15cm dish. Since different types of cells have diverse translation activities at different cell confluency, we recommend testing the harvest conditions and loading amount before your experiment.

  3. The optimal RNA amount in loading samples is 80-100 μg (sensitivity of 0.5). In cases where the RNA amount is lower than 80 μg, the sensitivity of the UV-Detector can be set to 0.2. We recommend using 200-600 μl of loading sample to get ideal and reproducible results.

  4. RNase R can digest mostlinear RNAs; however, some RNAs with highly structured 3' ends are resistant to RNase R treatment. In that case, a modified RNase R treatment with an additional polyadenylation step and replacement of K+ with Li+ in the reaction buffer can be used to deplete the linear RNAs (please see Xiao and Wilusz, 2019). In addition, the activity of RNase R varies between different batches; thus, we recommend checking the amount of enzyme and the reaction time before your experiments (Zhang, Y. et al., 2016).

Recipes

  1. 10× polysome buffer

    1 M KCl

    50 mM MgCl2·6H2O

    0.1 M HEPES (pH 7.4)

    Nuclease free water

  2. Wash buffer

    1× PBS

    100 μg/ml CHX

  3. Lysis buffer

    1× polysome buffer

    100 units/ml Ribonuclease inhibitor

    25 units/ml DNase I

    1× Protease Inhibitor cocktail (EDTA-free)

    2 mM DTT

    0.5% Sodium Deoxycholate

    0.5% Triton X-100

    100 μg/ml CHX

  4. 70% (w/v) sucrose solution

    70%(w/v) sucrose

    1× polysome buffer

    filtered with filter system

  5. 50% (w/v) sucrose grading solution

    71.4%(v/v) 70% sucrose solution

    1× polysome buffer

    100 units/ml Ribonuclease inhibitor

    1× Protease Inhibitor cocktail (EDTA-free)

    100 μg/ml CHX

  6. 10% (w/v) sucrose grading solution

    1.43% (v/v) 70% sucrose solution

    1× polysome buffer

    100 units/ml Ribonuclease inhibitor

    1× Protease Inhibitor cocktail (EDTA-free)

    100 μg/ml CHX

Acknowledgments

We want to thank all the members of Wang lab. This work was supported by the National Key Research and Development Program of China (MOST grant# 2018YFA0107602 to ZW) , the National Natural Science Foundation of China (NSFC grant #31730110, #91940303, and #32030064 to ZW , #31870814 to YY), the Strategic Priority Research Program of Chinese Academy of Sciences (grant #XDB38040100 to ZW andYY) and the Science and Technology Commission of Shanghai Municipality (STCSM grant # 19QA1410500 to YY). The present protocol was developed and applied in Yang et al. (2017).

Competing interests

The authors declare no competing financial interests.

References

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简介

[摘要]环状 RNA (circRNAs)是一种特殊类型的 RNA,没有 5'-和 3'-末端,广泛存在于真核生物中,已知可作为非编码 RNA 来调节基因表达,包括作为miRNA 海绵。最近的研究表明,许多外显子 circRNA 是由前体 mRNA 的反向剪接产生的,可以通过 IRES 或 m6A RNA 甲基化以与帽无关的方式进行翻译。然而,可翻译的circRNA的范围及其翻译产物在不同细胞和组织中的生物学功能仍不清楚。核糖体足迹ING 和蛋白质组学分析通常用于全局识别可翻译的 circRNA。然而,这两种方法具有灵敏度低由于circRNA特定的发现低效率读取或肽(即,背面拼接结是难以通过短恢复核糖体足迹的读取ING和蛋白质组分析的限制)。在这里,我们描述了一种使用多核糖体分析和 circRNA-seq 识别可翻译 circRNA 的替代方法。通常,与多核糖体相关的 RNA 是用蔗糖梯度分离的。然后多核糖体结合的circRNAs被富集的RNA酶ř处理,并通过鉴定配对末端深度测序。因此,该方法更灵敏比核糖体足迹和蛋白质组analys Ë小号为平移circRNAs的识别。


[背景]环状RNA小号(circRNA小号)是没有5'-和3'-端,其形成共价闭环一种特殊类型的RNA。他们是第一个发现的类病毒小号四十年前(桑格等,1976) 。最近,许多研究表明,circRNA广泛存在于真核生物中并以组织特异性方式表达(Rybak-Wolf等,2015;Chen,2020)。几种类型的circRNAs在细胞中被发现,包括外显子circRNA,EIciRNA ,和ciRNA (Kristensen的等人,2019。); 然而,它们中的大多数是通过前体 mRNA 的反向剪接产生的外显子 circRNA (Salzman等人,2012 年和 2013 年;Memczak等人,2013 年;Ashwal-Fluss等人,2014 年;Zhang , XO等人)人。,2014) 。因此,外显子最circRNAs序列是在相同的那些的线性的mRNA,和背面拼接结是用于circRNA检测独特的序列。
CircRNAs调节生物过程ES通过它们作为miRNA的海绵活动小号,蛋白质支架小号,和转录调节小号(Kristensen的等人。,2019) 。然而,大多数circRNA的功能仍不清楚。最近,几项研究表明,许多 circRNA 可以通过 IRES 或 m6A RNA 修饰的帽独立翻译进行翻译(Legnini等,2017;Pamudurti等,2017;Yang等,2017)。此外,一些circRNA编码肽报告来影响癌症增殖或寿命在FL IES (魏格特等人,2020;杨等人,2018;章,M.等人。,2018) ,这表明从circRNAs翻译产物发挥重要的生物学作用。然而,角色翻译circRNAs的是还是一个未知数。因此,翻译circRNAs的全基因组范围的识别是研究的重要途径IR的生物学功能。
通常使用两种不同类型的组学方法在 RNA(翻译组)和蛋白质(蛋白质组)水平上综合测量 mRNA 的翻译。核糖体足迹ING是一个新兴的技术,在全球范围内测量mRNA翻译通过核糖体保护基因片段的高通量测序; 然而,很难捕获翻译的 circRNA。由于核糖体保护的 mRNA 片段很短(大约 28-35nt),因此这种方法很容易遗漏反剪接点。蛋白质组学分析是一种通过酶消化肽的鉴定直接确定 mRNA 翻译产物的技术。相似于核糖体足迹ING ,通过背拼接结编码所述肽是难以IDENTIF ý通过串联质谱法。
我们开发了一种使用多核糖体分析和 circRNA-seq 识别可翻译 circRNA 的新方法。可以使用蔗糖梯度分离与 polysome 相关的 RNA,然后从不同的部分中分离。多核糖体结合的circRNAs由富集的RNA酶- [R处理和用于生成circRNA-SEQ库。得到的文库被双端测序,circRNAs通过反向剪接连接读数来识别。因此,我们的方法是更容易比,更灵敏的核糖体足迹和蛋白质组学analys Ë小号用于识别翻译circRNAs。

关键字:多核糖体剖析, circRNA 翻译, RNA-seq, 反向剪接点, RNase R

 
材料和试剂
 
10厘米15cm培养皿(Thermo Scientific的TM ,Nunc公司TM EasYDish,目录号小号:150464,150468 )
50毫升和15ml离心管中(Thermo Scientific的TM ,Thermo Scientific的TM的Nunc TM ,目录号小号:339650,339652)
1.5毫升和2ml Eppendorf管(无RNA酶)(CORNING,爱思进,目录号小号:MCT-150-C,MCT-200-C)
10毫升和25ml血清移液管(Thermo Scientific的TM ,Nunc公司TM ,目录号小号:170357N,170356N)
10 ml注射器(Sigma-Aldrich,目录号:Z248029)
移液针(BioComp,属于Gradient Master in Equipment)
25 cm细胞刮刀(JET BIOFIL,目录号:CSC011025)
聚丙烯离心管(Beckman coulter,目录号:331372)
真空过滤系统(Corning,目录号:430758)
细胞系:HEK293T
无核酸酶水(Sigma-Aldrich,目录号:W4502)
TRIzol TM试剂(Thermo Fisher,目录号:15596018)
氯仿(Sinoreagent,目录号:10006818)
异丙醇(Sinoreagent,目录号:80109218)
乙醇(Sigma-Aldrich,目录号:E7023)
Dulbecco改良鹰培养基(DMEM)(meilunbio,目录号:MA0212)
磷酸盐缓冲盐水(PBS)(Hyclone,目录号:SH30256.01)
核糖核酸酶抑制剂(Promega,目录号:N2518)
RQ1无RNA酶˚F REE DNA酶(Promega公司,目录号:M6101)
RNase R(Lucigen,目录号:RNR07250)
cOmplete TM EDTA-free Protease Inhibitor Cocktail(Roche,目录号:11873580001)
放线菌酮(CHX)(Sigma,目录号:C7698)
23.二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:43815)   
Triton TM X-100(Sigma-Aldrich,目录号:T8787)
溴酚蓝(Sigma-Aldrich,目录号:B0126)
带有 RiboErase(HMR)的KAPA Stranded RNA-Seq试剂盒(KAPA,目录号:KK8483)
RNA清洁和浓缩器试剂盒(ZYMO RESEARCH,目录号:R1019)
10× Polysome 缓冲液(见配方)
裂解缓冲液(见配方)
洗涤缓冲液(见配方)
70% 蔗糖溶液(见配方)
50% 蔗糖分级溶液(见配方)
10% 蔗糖分级溶液(见配方)
 
设备
 
超速离心机(带转子 SW41)(Beckman,型号:Optima XPN-100-IVD,目录号:A99846)
光谱仪(Thermo Fisher ,NanoDrop,目录号:ND-1000)
密度梯度分级分离系统(完整的系统包括管刺穿系统;注射器泵; UA-6检测器,带254级280nm的过滤器,R1馏分收集器,密度梯度流动池,电缆,和管道)(BRANDEL,目录号:BR-188)
Gradient Master (BioComp ,目录号:Biocomp 108)
 
软件
 
峰图(密度梯度分级系统的数据捕获软件,www.brandel.com)
TopHat 2/ TopHat-Fusion ( https://ccb.jhu.edu/software/tophat/index.shtml , https://ccb.jhu.edu/software/tophat/fusion_index.shtml )
CIRCexplorer2 ( https://circexplorer2.readthedocs.io/en/latest/ )
 
程序
 
蔗糖梯度的制备(图 1)
制备在不含核酸酶的水500毫升10×缓冲多聚核糖体和500ml的70%(W / V)在1×缓冲多核糖体的蔗糖溶液(见ř ecipes)。使用真空过滤系统过滤解决方案。
制备10%(W / V)和50%(W / V)蔗糖溶液70%(W / V)蔗糖溶液(见ř ecipes) 。
添加10%和50%蔗糖溶液,在该顺序,到在室温下在Beckman聚丙烯离心管中。
注:加半管先用 10% 蔗糖溶液,然后用针将50% 蔗糖溶液加到管底部的10% 溶液下。
加载在梯度主离心管以使一个10-50%(W / V)蔗糖梯度。蔗糖梯度可以立即使用或储存长期在-80℃(不超过2周)(小号EE注1)。
 
 
图1 。蔗糖梯度制备方案
 
样品制备
注意:使用前冷却所有溶液和缓冲液。如果可能,将样品一直放在冰上。
种子中的细胞10厘米15cm培养皿ES前一天实验(小号EE注2和表1 )。
 
表 1. 该协议的推荐时间表
 
在培养基中加入 100 μg/ml 放线菌酮,在 37 °C下孵育细胞10分钟。
丢弃培养基并用含有 100 µg/ml 放线菌酮的 10 ml 冰冷 PBS 清洗细胞两次。
向细胞中加入 10 ml 冰冷的 PBS,并用细胞刮刀收获细胞。
将细胞悬液转移到 15 ml 离心管中,并在4°C 下以 1,000 × g离心3 分钟。
去除上清液,将 250 μl 裂解缓冲液加入 15 ml 离心管中。
通过轻轻移液裂解细胞并将裂解物转移到 1.5 ml Eppendorf 管中。
将细胞裂解液在冰上放置 10分钟,然后在4°C 下以14,000 × g离心裂解液10 分钟以去除细胞碎片。
将上清液转移到新的 1.5 ml Eppendorf 管中。
稀1μl的样品用999微升水,并测量了使用近似RNA浓度一纳米d ROP光电分光计(小号EE注3)。
将样品放在冰上立即使用或立即将m冷冻在液氮中并储存在 -80 °C直至使用(不超过一个月)。
 
多聚体分析
除去的溶液的体积相同的的从顶部装载样品的10-50%蔗糖梯度,然后加载到样品的蔗糖梯度。最佳装载体积为200-500微升(小号EE注3)。保留 10% 的裂解物作为输入。
在 Beckman 超速离心机中使用 SW41 转子在4°C 下以 35,000 × g离心2.5小时。
在样品离心时设置密度梯度分馏系统。填充追溶液注射器[ 70%(W / V)与溴酚蓝蔗糖] ,冲洗管路,并校准紫外分光光度计按照设备指令小号。
轻轻地从转子除去管,并把它们成的密度梯度分馏系统。可替换地,让他们在4 ℃下直至使用(Ñ ø超过4小时)。
将样品加载到UA-6 检测器下并刺破管子底部,连接注射泵和检测器系统(见图 2)。
 
 
图 2. 密度梯度分级系统设置方案
 
为 2 ml Eppendorf 管编号并将它们放置在 R1 馏分收集器上。
注意:甲回合750微升样品的将在每个管收集。
将注射泵设置为 1.5 毫升/分钟的流速并打开远程模型。按时间设置馏分收集器。
使用峰值图表软件打开密度梯度分馏系统。记录与峰值图表(数据小号EE图3),并收集用级分收集器的部分。
将样品储存在 -80 °C直至使用(不超过一个月)。
 
 
图 3. Polysome 分析示例。代表 RNA 片段丰度的 OD254 (A254) 的吸光度由 UA-6 检测器测量。用密度梯度分级系统将蔗糖梯度分级为17个级分。A254 的值由峰图记录并如图所示绘制。
 
RNA-seq文库制备
根据制造商的说明加入750μl的TRIzol试剂到每个管中,并提取的RNA(Û本身氯仿,异丙醇,和70%的乙醇从样品和洗脱单独的RNA在约30μl的水)或存储在-80℃。
在自由的,monosome,光多核糖体或用等体积的,分别重多核糖体级分池RNA样品(例如,一个š如图3所示,级分1中称为“游离分数, ”分数6-7被称作“ monosome分数, ”级分8-10用等体积合并的小号和称为“光多核糖体级分,”和分数11-16合并用等体积的和称为“重馏分多核糖体” )。
使用 Nano D rop测量每种 RNA 混合物的 RNA 浓度。
在 37°C 下用 DNase I(无 RNase)处理 RNA 15 分钟。
2 微克 RNA x 微升
DNase I 1μl
10×DNase I 缓冲液 5 μl
水 44-x μl
清理使用的RNA的根据生产商的说明书ZYMO RNA清洁和浓缩试剂盒小号。
治疗所述DNA酶I处理过的用RNaseř的RNA在37℃下30分钟(小号EE注4)。
1 微克 RNA x 微升
RNase R 1 微升
10×RNase R 缓冲液 5 μl
水 44-x μl
清理使用的RNA的根据生产商的说明书ZYMO RNA清洁和浓缩试剂盒(ê luted在20μl水中的RNA) 。
根据制造商的说明,使用带有 RiboErase的KAPA 链式 RNA-seq 试剂盒构建 RNA-seq 库。
序列使用的库一个与150bp的配对末端方法一个每个样品亿阅读深度。
注:我们建议Illumina的HiSeq ® 3000 / HiSeq 4000测序系统的RNA-seq的。
 
鉴定可翻译的circRNA
对原始测序读数进行质量控制并修剪适配器序列。丢掉低-品质的读取。从读取中修剪适配器序列并将m映射到基因组。
使用 TopHat2 将读数与参考基因组对齐,并使用 TopHat-Fusion 映射未映射的读数。
使用CIRCexplorer2 管道注释和组装 circRNA 转录本(参见 GitHub 中的详细信息:https ://circexplorer2.readthedocs.io/en/latest/ )(Zhang ,XO等,2016)。
 
笔记
 
如果没有 Gradient Master 进行梯度形成,我们建议使用另一种方法通过冻结和解冻来形成梯度。为了使一个10-50%(W / V)蔗糖梯度,制备50%(W / V),40%(W / V),30%(W / V),20%(W / V) ,和10 % (w/v) 蔗糖溶液s 。放2毫升的50%的蔗糖到一个贝克曼离心机管和冻结干冰-乙醇,然后把40%的溶液(2ml)中和也冻结,并重复与其他解决方案。所述管可以是冷冻和储存d在-80℃下。使用前一天,将解冻的冷藏库的管道(4℃)过夜,一个第二它们是准备使用。
牛逼Ø保持与活动翻译的细胞,它是更好的收获米约为70-80%汇合。例如,对于 HEK293T,细胞可以在实验前一天接种在 10 cm(2.2 × 10 6 个细胞)或 15 cm(5.0 × 10 6 个细胞)培养皿中,并在大约 7.0 × 10 6 个细胞/10 cm培养皿中收获或16.0 × 10 6 个细胞/15cm 培养皿。由于不同类型的细胞具有不同的细胞融合多样的翻译活动,我们建议测试收获条件小号实验之前和负载量。
上样样品的最佳 RNA 量为 80-100 μg(灵敏度为 0.5)。万一š其中所述RNA量低于80微克,灵敏度的UV检测器可以被设置为0.2。我们建议您使用200-600微升装载样品的获得理想的和可重复的结果小号。
RNase R 可以消化大多数线性 RNA ;然而,一些具有高度结构化 3' 末端的 RNA 对 RNase R 处理具有抗性。在这种情况下,与K的额外多聚腺苷酸化步骤和替换经修饰的核糖核酸酶ř治疗+与Li +在所述反应缓冲液可用于耗尽线性的RNA(请参阅萧和Wilusz,2019) 。另外,不同批次的RNase R活性不同;因此,我们建议您在实验前检查酶的量和反应时间(Zhang , Y. et al ., 2016) 。
 
食谱
 
10×polysome 缓冲液
1 M氯化钾
50 mM MgCl 2 ·6H 2 O
0.1 M HEPES (pH 7.4)
无核酸酶水
洗涤缓冲液
1×PBS
100 微克/毫升 CHX
裂解缓冲液
1×polysome 缓冲液
100 单位/毫升核糖核酸酶抑制剂
25 单位/ml DNase I
1× 蛋白酶抑制剂混合物(不含 EDTA)
2 毫米 DTT
0.5%脱氧胆酸钠
0.5% 海卫 X-100
100 微克/毫升 CHX
70% (w/v) 蔗糖溶液
70%(w/v) 蔗糖
1×polysome 缓冲液
用过滤系统过滤
50% (w/v) 蔗糖分级溶液
71.4%(v/v) 70% 蔗糖溶液
1×polysome 缓冲液
100 单位/毫升核糖核酸酶抑制剂
1× 蛋白酶抑制剂混合物(不含 EDTA)
100 微克/毫升 CHX
10% (w/v) 蔗糖分级溶液
1.43% (v/v) 70% 蔗糖溶液
1×polysome 缓冲液
100 单位/毫升核糖核酸酶抑制剂
1× 蛋白酶抑制剂混合物(不含 EDTA)
100 微克/毫升 CHX
 
致谢
我们要感谢王实验室的所有成员。这项工作得到了中国国家重点研发计划(MOST grant# 2018YFA0107602 to ZW)、国家自然科学基金(NSFC grant #31730110, #91940303, and #32030064 to ZW, #31870814 to YY)的支持, 中国科学院战略重点研究计划(ZW 和YY 授予#XDB38040100)和上海市科委(STCSM 授予#19QA1410500 到YY)。本协议是在杨等人中开发和应用的。(2017)。
 
利益争夺
 
作者声明没有相互竞争的经济利益。
 
参考
 
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引用:Ye, Y., Wang, Z. and Yang, Y. (2021). Comprehensive Identification of Translatable Circular RNAs Using Polysome Profiling. Bio-protocol 11(18): e4167. DOI: 10.21769/BioProtoc.4167.
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