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RNA Chromatin Immunoprecipitation (RNA-ChIP) in Caenorhabditis elegans
秀丽隐杆线虫的RNA染色质免疫共沉淀(RNA-ChIP)   

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Abstract

The RNA chromatin immunoprecipitation assay (RNA-ChIP) allows detection and quantification of RNA–protein interactions using in vivo cross-linking with formaldehyde followed by immunoprecipitation of the RNA–protein complexes. Here we describe the RNA–ChIP protocol that we have adapted for Caenorhabditis elegans (C. elegans) to detect interaction between the nuclear Argonaute CSR-1 (chromosome segregation and RNAi deficient) protein and its target nascent RNAs. We have used a transgenic strain expressing a recombinant long isoform of CSR-1 protein fused with N-terminal 3x FLAG epitope.

Materials and Reagents

  1. Escherichia coli (E. coli) OP50 [available from the Caenorhabditis Genetics Center (CGC)]
  2. Anti–FLAG M2 affinity gel (Sigma-Aldrich, catalog number: A2220 )
  3. 1-Bromo-3-chloropropane (BCP) (Sigma-Aldrich, catalog number: B9673 )
  4. 1 M CaCl2 solution (Sigma-Aldrich, catalog number: 21114 )
  5. Distilled water (DNase/RNase-free UltraPureTM) (Life Technologies, InvitrogenTM, catalog number: 10977-015 )
  6. 10 mM dNTP mix (Thermo Fisher Scientific, catalog number: R0191 )
  7. EDTA (0.5 M, pH 8.0) UltraPureTM (Life Technologies, InvitrogenTM, catalog number: 15575-020 )
  8. Ethanol 200 proof (for molecular biology) (Sigma-Aldrich, catalog number: E7023 )
  9. FLAG peptide (Sigma-Aldrich, catalog number: F4799 )
  10. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  11. GlycoBlueTM coprecipitant (15 mg/ml) (Life Technologies, Ambion®, catalog number: AM4530)
  12. 100x halt protease inhibitor cocktail (Thermo Fisher Scientific, catalog number: 78430 )
  13. Isopropanol (2-propanol, for molecular biology) (Sigma-Aldrich, catalog number: I9516 )
  14. KH2PO4 (Sigma-Aldrich, catalog number: P0662 )
  15. 8 M LiCl solution (Sigma-Aldrich, catalog number: L7026 )
  16. Maxima reverse transcriptase (Thermo Fisher Scientific, catalog number: EP0741 )
  17. 1 M MgCl2 solution (Sigma-Aldrich, catalog number: M1028 )
  18. 1 M MgSO4 solution (Sigma-Aldrich, catalog number: M3409 )
  19. Na2HPO4 (Sigma-Aldrich, catalog number: S3264 )
  20. 5 M NaCl solution (Life Technologies, Ambion®, catalog number: AM9759 )
  21. NP-40 [10% (w/v) aqueous solution] (Pierce Antibodies, catalog number: 85124 )
  22. Paraformaldehyde (32% solution, EM grade) (VWR International, catalog number: 15714-S )
  23. Phase lock gel heavy (1.5 ml) (5PRIME, catalog number: 2302810 )
  24. Phenol:Chloroform:IAA (25:24:1, pH 6.6) (Life Technologies, Ambion®, catalog number: AM9730 )
  25. Proteinase K (~20 mg/ml) (recombinant) (PCR grade) (Thermo Fisher Scientific, catalog number: EO0491 )
  26. 2x QuantiFast SYBR® Green qPCR master mix (QIAGEN, catalog number: 204154 )
  27. Random hexamers (100 µM) (Thermo Fisher Scientific, catalog number: SO142 )
  28. RNase inhibitor SUPERase InTM (20 U/μl) (Life Technologies, Ambion®, catalog number: AM2694 )
  29. 20% SDS solution (Life Technologies, Ambion®, catalog number: AM9820 )
  30. 3 M sodium acetate (pH 5.5) solution (Life Technologies, Ambion®, catalog number: AM9740 )
  31. Sodium deoxycholate stock solution [10% (w/v) in distilled water] (Pierce Antibodies, catalog number: 89904 )
  32. TRI Reagent® solution (Life Technologies, Ambion®, catalog number: AM9738 )
  33. 1 M Tris-HCl (pH 7.5) UltraPureTM (Life Technologies, InvitrogenTM, catalog number: 15567-027 )
  34. Triton-X 100 detergent solution (Pierce Antibodies, catalog number: 85111 )
  35. The sequence of primers used in Figure 1 to detect mes-4 pre-mRNA: Forward GGATACATCAATGGAGAAATGGA (spanning exon 2 and intron 3) and Reverse ACAACTCGCGTGAAATTTACTAC (spanning intron 3)
    Note: DNA oligos has been synthesized by Integrated DNA Technologies (IDT).
  36. 1x M9 buffer (see Recipes)
  37. Nuclei extraction buffer (see Recipes)
  38. RIPA buffer (see Recipes)
  39. TSE 150 buffer (see Recipes)
  40. TSE 500 buffer (see Recipes)
  41. TSE 1,000 buffer (see Recipes)
  42. LiCl buffer (see Recipes)
  43. TE buffer (see Recipes)
  44. Elution buffer (see Recipes)

Equipment

  1. 1.5 ml Bioruptor TPX® polymethylpentene tubes (Diagenode, catalog number: M500-50 )
  2. Microfuge tubes (1.5 ml, RNase free) (Life Technologies, InvitrogenTM, catalog number: AM12400 )
  3. Bioruptor® standard UCD-200 sonicator (Diagenode, catalog number: B01010003 )
  4. Dura-GrindTM stainless steel dounce tissue grinder 7 ml size (Wheaton Scientific, catalog number: 357572 )
  5. Real-time PCR machine (e.g. Eppendorf, Mastercycler® ep realplex4)
  6. Refrigerated tabletop microcentrifuge
  7. LabquakeTM tube rotators (Thermo Fisher Scientific, catalog number: 400220Q )
  8. Vortex

Procedure

  1. Fixation and nuclei extraction
    1. Grow synchronized populations of worms on one large LB agar plate (100 x 15 mm) seeded with OP50 bacteria (~20,000 worms).
    2. Wash worms off the plate with cold M9.
    3. Centrifuge for 1 min at 1,200 x g to pellet the worms.
    4. Wash 3 times with cold M9.
    5. Centrifuge for 1 min at 1,200 x g to pellet the worms.
    6. Fix the worms with 2% paraformaldehyde in 10 ml M9 buffer for 30 min at room temperature on rotation.
    7. Centrifuge for 1 min at 1,200 x g to pellet the worms.
    8. Discard the supernatant and wash one time with 10 ml cold 0.1 M Tris-HCl (pH 7.5).
    9. Centrifuge for 1 min at 1,200 x g to pellet the worms.
    10. Wash the worm pellet twice with 10 ml cold M9 buffers.
    11. Centrifuge for 1 min at 1,200 x g to pellet the worms and discard the supernatant.
      Note: At this step you can freeze the worm pellet and store at -80 °C.
    12. Resuspend worm pellet in 2 ml cold Nuclei extraction buffer.
    13. Transfer 2 ml of worms to a steel dounce (on ice) and stroke at least 30 times.
      Note: Check under the microscope that the worms have been crushed.
    14. Transfer 2 ml of lysate to two RNase free 1.5 ml microfuge tubes and centrifuge 2 min at 40 x g at 4 °C.
    15. Transfer the supernatant to two new RNase free 1.5 ml microfuge tubes.
    16. Centrifuge for 5 min at 1,000 x g at 4 °C to pellet the nuclear fraction.
    17. Discard the supernatant and wash the nuclear pellet with 1 ml of cold nuclei extraction buffer.
      Note: Combine the two pellets in one RNase free 1.5 ml microfuge tube.
    18. Centrifuge for 5 min at 1,000 x g at 4 °C to pellet the nuclear fraction.
    19. Repeat steps 17 and 18 twice.
    20. Centrifuge for 5 min at 1,000 x g at 4 °C to pellet the nuclear fraction.
    21. Resuspend the nuclear pellet in 500 μl RIPA buffer.

  2. Sonication
    1. Transfer the lysate to a 1.5 ml Bioruptor TPX® polymethylpentene tube.
    2. Sonicate sample for 20 min at 4 °C (settings: 30 sec on, 30 sec off, level high 320 W, wave frequency 20 kHz).
    3. Centrifuge for 5 min at 12,000 x g at 4 °C. Transfer the supernatant (containing chromatin) to a new RNase free 1.5 ml microfuge tube.
      Note: At this step samples can be stored at -80 °C.

  3. Immunoprecipitation
    1. Take 1/20 fraction of the supernatant for the input RNA extraction and store on ice.
    2. Use 30 μl of anti-Flag M2 affinity gel for each immunoprecipitation.
    3. Equilibrate the affinity gel with 1 ml of RIPA buffer.
    4. Centrifuge for 1 min at 1,000 x g at 4 °C.
    5. Repeat steps 27 and 28.
    6. Add supernatant from step 24 to the equilibrated affinity gel.
    7. Incubate on rotation (continuous rotation at 8rpm) for 1 h at 4 °C.
    8. Centrifuge for 1 min at 1,000 x g at 4 °C.
    9. Wash the affinity gel with 1 ml TSE150 buffer and incubate on rotation for 1 min at 4 °C.
    10. Centrifuge for 1 min at 1,000 x g at 4 °C.
    11. Discard the supernatant and wash the affinity gel 2 times with 1 ml TSE500 buffer and incubate on rotation for 1 min at 4 °C.
    12. Centrifuge for 1 min at 1,000 x g at 4 °C.
    13. Discard the supernatant and wash the affinity gel with 1 ml TSE1000 buffer and incubate on rotation for 5 min at 4 °C.
    14. Centrifuge for 1 min at 1,000 x g at 4 °C.
    15. Discard the supernatant and wash the affinity gel with 1 ml LiCl buffer and incubate on rotation for 5 min at 4 °C.
    16. Centrifuge for 1 min at 1,000 x g at 4 °C.
    17. Discard the supernatant and wash the affinity gel 2 times with 1 ml TE buffer.
    18. Centrifuge for 1 min at 1,000 x g at 4 °C.
    19. Discard the supernatant and remove as much as possible buffer using P200 pipette tips.
    20. Elute the protein-RNA complexes from the affinity gel using 200 μl of elution buffer.
    21. Incubate with rotation for 1 h at 4 °C.
    22. Centrifuge for 1 min at 1,000 x g at 4 °C.
    23. Transfer the supernatant to a new RNase free 1.5 ml microfuge tube.

  4. Reverse crosslink
    1. Take the 200 μl of supernatant from step 47 and input RNA from step 25 (to a final volume of 200 μl with elution buffer) and treat them with 1 μl (20 μg) of proteinase K for 1 h at 45 °C.

  5. RNA purification
    1. Add 750 μl of TRI Reagent® solution to the samples from step 48 and vortex for 30 sec.
    2. Add 100 μl of BCP and vortex for 30 sec.
    3. Incubate at room temperature for 10 min.
    4. Centrifuge for 15 min at 12,000 x g at 4 °C.
    5. Transfer the top aqueous phase in a new RNase free 1.5 ml microfuge tube.
    6. Add 1.5 μl of GlycoblueTM and 50 μl of 3 M sodium acetate.
    7. Mix and add 1 volume of isopropanol (typically 400-500 μl).
    8. Incubate at room temperature for 5 min.
    9. Centrifuge for 20 min at 12,000 x g at 4 °C.
    10. Wash the RNA pellet with 1 ml of 70% ethanol.
    11. Centrifuge for 5 min at 7,500 x g at 4 °C.
    12. Resuspend the pellet in 44 μl of RNase-free UltraPureTM distilled water.

  6. DNase treatment
    1. Add 5 μl of 10x TURBO DNase buffer and 1 μl of TURBO DNase to the samples from step 60.
    2. Incubate 30 min at 37 °C.
    3. Add 450 μl of RNase-Free UltraPureTM distilled water (total volume 500 μl).
    4. Add 500 μl of Phenol:Chloroform:IAA and mix by vortex.
    5. Transfer the mixture to 1.5 ml Phase Lock Gel tube and centrifuge at 12,000 x g for 5 min at 4 °C.
    6. Transfer top aqueous phase in a new RNase free 1.5 ml microfuge tube.
    7. Add 1.5 μl of GlycoblueTM and 50 μl of 3 M sodium acetate and mix by vortex.
    8. Add 1 volume of isopropanol (typically 400-500 μl) and mix by vortex.
    9. Incubate at room temperature for 5 min.
    10. Centrifuge for 20 min at 12,000 x g at 4 °C.
    11. Wash the RNA pellet with 1 ml of 70% ethanol.
    12. Centrifuge for 5 min at 7,500 x g at 4 °C.
    13. Resuspend in 12.5 μl ofRNase-Free UltraPureTM distilled water.

  7. cDNA Synthesis
    1. Add to the samples from step 73, 1 μl of random hexamer primers, 4 μl of 5x reaction buffer, 0.5 μl of RNase Inhibitor, 1 μl of dNTP mix (0.5 mM final concentration), and 1 μl of Maxima reverse transcriptase.
    2. Incubate 10 min at 25 °C followed by 60 min at 37 °C.
    3. Stop the reaction by heating at 70 °C for 10 min.
      Note: The reverse transcription reaction can be stored at -20 °C.

  8. qPCR
    1. Dilute the 20 μl sample from step 76 with 100 μl of nuclease free water and use 5 μl as a template in 25 μl qPCR reaction (performed in triplicate reactions); add 12.5 μl of 2x QuantiFast SYBR® Green mix, and primers to a final concentration of 0.6 μM. For the amplification of nascent RNAs, the primers have to be designed in such a way that at least one PCR primer (forward or reverse) is located in an intron of a target gene and the amplicon is 70-100 bp long. The average Ct value from triplicate qPCR reactions will be consider to calculate the percentage of input. For more information about the percentage of input calculation see http://www.lifetechnologies.com/us/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html.
    2. For qPCR cycling conditions use the parameters suggested by QuantiFast SYBR® Green mix manufacturers.
      Note: The best way to represent the data is by percentage of input (see Figure 1).

Representative data



Figure 1. RNA–ChIP qPCR results obtained with an antibody against FLAG epitope and nuclear extracts from transgenic worms expressing FLAG:CSR-1 protein and control non-transgenic worms (no FLAG). The primers used in a qPCR assay have been designed to span one intron of the pre-mRNA of a known target transcript of CSR-1. The results from three independent experiments (biological replicates) are shown as percentage of input. Error bars represent the standard deviation.

Notes

The protocol has been applied to the transgenic strain expressing a recombinant CSR-1 protein fused with Flag epitope. However, it can be used with virtually any Flag-tagged nuclear proteins expressed in C. elegans. Moreover, if a primary antibody raised against the protein of interest is available, it can be crosslinked to Protein A/G resin and used instead of the anti-Flag M2 affinity gel. In this case, it is recommended to choose an antibody that has been tested in ChIP experiments. Because of the high background of the RNA-ChIP assay, it is highly recommended to always use a non-transgenic strain as a control in each experiment or knock out strains if primary antibody raised against the protein of interest will be used. The following protocol can also be used for cytoplasmic RNA-binding proteins. In this case, the nuclear extraction step can be omitted.

Recipes

  1. 1x M9 buffer
    3 g KH2PO4
    6 g Na2HPO4
    17 ml 5 M NaCl
    1 ml 1 M MgSO4 in 1 L of deionized water
  2. Nuclei extraction buffer
    10 mM Tris-HCl (pH 7.5)
    2 mM MgCl2
    3 mM CaCl2
    0.5% NP-40
    10% glycerol
    100 U/ml RNase inhibitor
    1x protease inhibitor cocktails
  3. RIPA buffer
    0.1% SDS
    0.1% deoxycholate
    1% Triton X-100
    1 mM EDTA
    10 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    100 U/ml RNase inhibitor, protease inhibitor cocktails
  4. TSE 150 buffer
    0.1% SDS
    1% Triton X-100
    2 mM EDTA
    20 mM Tris-HCl (pH 7.5)
    150 mM NaCl
    100 U/ml RNase inhibitor
  5. TSE 500 buffer
    Same as TSE 150 but with 500 mM NaCl
  6. TSE 1,000 buffer
    Same as TSE 150 but with 1 M NaCl
  7. LiCl buffer
    250 mM LiCl
    1% NP40
    1% deoxycholate
    1 mM EDTA
    10 mM Tris-HCl (pH 7.5)
    100 U/ml RNase inhibitor
  8. TE buffer
    10 mM Tris-HCl (pH 7.5)
    1 mM EDTA
  9. Elution buffer
    10 mM Tris-HCl (pH 7.5)
    0.5% NP-40
    200 mM NaCl
    300 μg/ml FLAG peptide
    100 U/ml RNase inhibitor

Acknowledgments

The transgenic strain expressing a recombinant CSR-1 protein fused with 3x FLAG epitope was kindly provided by C. Mello (University of Massachusetts, Worcester). This work was supported by US National Institutes of Health grant 1DP2OD006412-01 (A. G.). This protocol has been adapted from our previous work (Cecere et al., 2014).

References

  1. Bittencourt, D. and Auboeuf, D. (2012). Analysis of co-transcriptional RNA processing by RNA-ChIP assay. Methods Mol Biol 809: 563-577.
  2. Cecere, G., Hoersch, S., O'Keeffe, S., Sachidanandam, R. and Grishok, A. (2014). Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape. Nat Struct Mol Biol 21(4): 358-365.

简介

RNA染色质免疫沉淀测定(RNA-ChIP)允许使用体内与甲醛交联,随后RNA-蛋白复合物的免疫沉淀来检测和定量RNA-蛋白质相互作用。 在这里我们描述我们已经适应于秀丽隐杆线虫( C. elegans )的RNA-ChIP协议以检测核Argonaute CSR-1(染色体分离和RNAi缺陷 )蛋白及其目标新生RNA。 我们使用表达与N-末端3x FLAG表位融合的CSR-1蛋白的重组长同种型的转基因菌株。

材料和试剂

  1. 大肠杆菌(大肠杆菌)OP50 [可从Caenorhabditis 遗传中心(CGC)获得]
  2. 抗FLAG M2亲和凝胶(Sigma-Aldrich,目录号:A2220)
  3. 1-溴-3-氯丙烷(BCP)(Sigma-Aldrich,目录号:B9673)
  4. 1M CaCl 2溶液(Sigma-Aldrich,目录号:21114)
  5. 蒸馏水(DNase/RNase-free UltraPure TM)(Life Technologies,Invitrogen TM,目录号:10977-015)
  6. 10mM dNTP混合物(Thermo Fisher Scientific,目录号:R0191)
  7. EDTA(0.5M,pH 8.0)UltraPure TM(Life Technologies,Invitrogen TM,目录号:15575-020)
  8. 乙醇200标准(用于分子生物学)(Sigma-Aldrich,目录号:E7023)
  9. FLAG肽(Sigma-Aldrich,目录号:F4799)
  10. 甘油(Sigma-Aldrich,目录号:G5516)
  11. GlycoBlue TM共沉淀(15mg/ml)(Life Technologies,Ambion ,目录号:AM4530)
  12. 100x暂停蛋白酶抑制剂混合物(Thermo Fisher Scientific,目录号:78430)
  13. 异丙醇(2-丙醇,分子生物学)(Sigma-Aldrich,目录号:I9516)
  14. KH sub 2 PO 4(Sigma-Aldrich,目录号:P0662)
  15. 8 M LiCl溶液(Sigma-Aldrich,目录号:L7026)
  16. Maxima逆转录酶(Thermo Fisher Scientific,目录号:EP0741)
  17. 1M MgCl 2溶液(Sigma-Aldrich,目录号:M1028)
  18. 1M MgSO 4溶液(Sigma-Aldrich,目录号:M3409)
  19. Na 2 HPO 4(Sigma-Aldrich,目录号:S3264)
  20. 5 M NaCl溶液(Life Technologies,Ambion ,目录号:AM9759)
  21. NP-40 [10%(w/v)水溶液](Pierce Antibodies,目录号:85124)
  22. 多聚甲醛(32%溶液,EM级)(VWR International,目录号:15714-S)
  23. 锁相凝胶重(1.5ml)(5PRIME,目录号:2302810)
  24. 苯酚:氯仿:IAA(25:24:1,pH 6.6)(Life Technologies,Ambion ,目录号:AM9730)
  25. 蛋白酶K(〜20mg/ml)(重组)(PCR级)(Thermo Fisher Scientific,目录号:EO0491)
  26. 2x QuantiFast SYBR Green qPCR主混合物(QIAGEN,目录号:204154)
  27. 随机六聚体(100μM)(Thermo Fisher Scientific,目录号:SO142)
  28. 核酸酶抑制剂SUPERase In (20U /μl)(Life Technologies,Ambion ,目录号:AM2694)
  29. 20%SDS溶液(Life Technologies,Ambion ,目录号:AM9820)
  30. 3 M乙酸钠(pH 5.5)溶液(Life Technologies,Ambion ,目录号:AM9740)
  31. 脱氧胆酸钠储备液[10%(w/v)在蒸馏水中](Pierce Antibodies,目录号:89904)
  32. TRI Reagent 溶液(Life Technologies,Ambion ,目录号:AM9738)
  33. 1M Tris-HCl(pH7.5)UltraPure TM(Life Technologies,Invitrogen TM ,目录号:15567-027)
  34. Triton-X 100洗涤剂溶液(Pierce Antibodies,目录号:85111)
  35. 图1中用于检测mes-4前mRNA的引物序列:正向GGATACATCAATGGAGAAATGGA(跨越外显子2和内含子3)和反向ACAACTCGCGTGAAATTTACTAC(跨越内含子3)
    注意:DNA oligo已由Integrated DNA Technologies(IDT)合成。
  36. 1x M9缓冲区(参见配方)
  37. 核提取缓冲液(参见配方)
  38. RIPA缓冲区(参见配方)
  39. TSE 150缓冲区(参见配方)
  40. TSE 500缓冲液(见配方)
  41. TSE 1,000缓冲区(参见配方)
  42. LiCl缓冲液(见配方)
  43. TE缓冲区(参见配方)
  44. 洗脱缓冲液(见配方)

设备

  1. 1.5ml Bioruptor TPX 聚甲基戊烯管(Diagenode,目录号:M500-50)
  2. 微量离心管(1.5ml,不含RNase)(Life Technologies,Invitrogen TM,目录号:AM12400)
  3. Bioruptor 标准UCD-200超声波仪(Diagenode,目录号:B01010003)
  4. Dura-Grind TM 不锈钢杜松组织研磨机7ml(Wheaton Scientific,目录号:357572)
  5. 实时PCR仪(例如 Eppendorf,Mastercycler ep realplex 4
  6. 制冷台式微量离心机
  7. Labquake 管旋转器(Thermo Fisher Scientific,目录号:400220Q)
  8. 涡流

程序

  1. 固定和核提取
    1. 在一个接种有OP50细菌(约20,000个蠕虫)的大LB琼脂平板(100×15mm)上生长同步的蠕虫群体。
    2. 用冷的M9清洗板上的蠕虫。
    3. 在1,200×g离心1分钟,以使蠕虫沉淀
    4. 用冷M9洗涤3次。
    5. 在1,200×g离心1分钟,以使蠕虫沉淀
    6. 固定蠕虫与2%多聚甲醛在10ml M9缓冲液中30分钟,在室温下旋转。
    7. 在1,200×g离心1分钟,以使蠕虫沉淀
    8. 弃去上清液,并用10ml冷的0.1M Tris-HCl(pH 7.5)洗涤一次
    9. 在1,200×g离心1分钟,以使蠕虫沉淀
    10. 用10毫升冷M9缓冲液清洗蜗杆沉淀两次。
    11. 在1,200×g离心1分钟以沉淀蠕虫并弃去上清液。
      注意:在这一步,你可以冻结蠕虫丸,并存储在-80°C。
    12. 将蠕虫颗粒在2ml冷核提取缓冲液中重悬
    13. 转移2毫升蠕虫到钢dounce(冰)和中风至少30次。
      注意:在显微镜下检查蠕虫是否被破坏。
    14. 转移2毫升裂解物到两个无RNA酶的1.5毫升微量离心管和离心2分钟,在40℃,4℃。
    15. 将上清转移到两个新的无RNase的1.5ml微量离心管中
    16. 在4℃下以1,000×g离心5分钟以沉淀核部分。
    17. 弃去上清液并用1ml冷核提取缓冲液洗涤核沉淀。
      注意:在一个不含RNA酶的1.5ml微量离心管中混合两种沉淀。
    18. 在4℃下以1,000×g离心5分钟以沉淀核部分。
    19. 重复步骤17和18两次
    20. 在4℃下以1,000×g离心5分钟以沉淀核部分。
    21. 重悬在500μlRIPA缓冲液中的核沉淀。

  2. 超声
    1. 将裂解物转移到1.5ml Bioruptor TPX 聚甲基戊烯管中。
    2. 在4℃下超声处理样品20分钟(设置:30秒开,30秒关,水平高320W,波频率20kHz)。
    3. 在4℃下以12,000×g离心5分钟。 转移 上清液(含有染色质)至新的无RNA酶的1.5ml微量离心机 管 注意:在这一步,样品可以储存在-80°C。

  3. 免疫沉淀
    1. 取1/20部分的上清液用于输入RNA提取并储存在冰上
    2. 每次免疫沉淀使用30μl的抗Flag M2亲和凝胶
    3. 用1ml RIPA缓冲液平衡亲和凝胶
    4. 在4℃下以1000xg离心1分钟。
    5. 重复步骤27和28。
    6. 将步骤24的上清液加入到平衡的亲和凝胶中
    7. 在4℃下孵育1小时(以8rpm连续旋转)
    8. 在4℃下以1000xg离心1分钟。
    9. 用1ml TSE150缓冲液洗涤亲和凝胶,并在4℃下旋转孵育1分钟
    10. 在4℃下以1000xg离心1分钟。
    11. 弃去上清液,用1ml洗涤亲和凝胶2次   TSE500缓冲液中,并在4℃下旋转孵育1分钟。
    12. 在4℃下以1000xg离心1分钟。
    13. 弃去上清液,用1ml TSE1000缓冲液洗涤亲和凝胶,并在4℃下旋转孵育5分钟。
    14. 在4℃下以1000xg离心1分钟。
    15. 弃去上清液并用1ml LiCl缓冲液洗涤亲和凝胶,并在4℃下旋转孵育5分钟。
    16. 在4℃下以1000xg离心1分钟。
    17. 弃去上清液,用1ml TE缓冲液洗涤亲和凝胶2次
    18. 在4℃下以1000xg离心1分钟。
    19. 弃去上清液,并使用P200移液器吸头尽可能多的缓冲液。
    20. 使用200μl洗脱缓冲液从亲和凝胶洗脱蛋白质-RNA复合物
    21. 在4℃下旋转孵育1小时。
    22. 在4℃下以1000xg离心1分钟。
    23. 转移上清液到一个新的无RNA酶1.5毫升微量离心管。

  4. 反向交联
    1. 取来自步骤47的200μl上清液,并输入来自步骤25的RNA (用洗脱缓冲液至终体积为200μl)并用1处理 μl(20μg)蛋白酶K在45℃下孵育1小时。

  5. RNA纯化
    1. 向步骤48的样品中加入750μlTRI试剂溶液并涡旋30秒。
    2. 加入100μlBCP,涡旋30秒
    3. 在室温下孵育10分钟。
    4. 在4℃下以12,000xg离心15分钟。
    5. 将顶部水相转移到新的无RNA酶的1.5ml微量离心管中
    6. 加入1.5μlGlycoblue TM 和50μl的3M乙酸钠
    7. 混合并加入1体积的异丙醇(通常为400-500μl)
    8. 在室温下孵育5分钟
    9. 在4℃下以12,000xg离心20分钟。
    10. 用1 ml 70%乙醇洗涤RNA沉淀
    11. 在4℃下以7,500×g离心5分钟。
    12. 将沉淀重悬于44μl无RNase的UltraPure TM蒸馏水中。

  6. DNase治疗
    1. 向步骤60的样品中加入5μl10x TURBO DNase缓冲液和1μlTURBO DNase。
    2. 在37℃孵育30分钟。
    3. 加入450μl无RNase-Free UltraPure TM 蒸馏水(总体积500μl)。
    4. 加入500μl的苯酚:氯仿:IAA,涡旋混合
    5. 将混合物转移到1.5ml Phase Lock Gel管中并在4℃下以12,000×g离心5分钟。
    6. 将顶层水相转移到新的无RNA酶的1.5ml微量离心管中
    7. 加入1.5μlGlycoblue TM 和50μl的3M乙酸钠,并涡旋混合。
    8. 加入1倍体积的异丙醇(通常为400-500μl),并通过涡旋混合
    9. 在室温下孵育5分钟
    10. 在4℃下以12,000xg离心20分钟。
    11. 用1 ml 70%乙醇洗涤RNA沉淀
    12. 在4℃下以7,500×g离心5分钟。
    13. 重悬于12.5μl无RNA酶UltraPure TM蒸馏水中。

  7. cDNA合成
    1. 加入来自步骤73的样品,1μl随机六聚体引物,4μl   5x反应缓冲液,0.5μlRNase抑制剂,1μldNTP混合物(0.5mM   最终浓度)和1μlMaxima逆转录酶
    2. 在25℃孵育10分钟,然后在37℃孵育60分钟
    3. 通过在70℃加热10分钟停止反应 注意:逆转录反应可以储存在-20℃。

  8. qPCR
    1. 用100μl无核酸酶的水稀释步骤76的20μl样品 并在25μlqPCR反应中使用5μl作为模板(进行 一式三份反应);加入12.5μl的2x QuantiFast SYBR 绿色混合物,和  引物至终浓度0.6μM。对于放大 新生RNA,引物必须以这样的方式设计: 至少一个PCR引物(正向或反向)位于a的内含子中 靶基因,并且扩增子的长度为70-100bp。平均Ct值 从一式三份qPCR反应中将考虑计算 输入百分比。有关输入百分比的详细信息 计算参见 http://www.lifetechnologies.com/us/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html 。
    2. 对于qPCR循环条件,使用QuantiFast SYBR ®绿色混合制造商建议的参数。
      注意:表示数据的最佳方法是使用输入的百分比(见图1)。

代表数据



图1.使用针对FLAG表位的抗体和来自表达FLAG:CSR-1蛋白和对照非转基因蠕虫(无FLAG)的转基因蠕虫的核提取物的抗体获得的RNA-ChIP qPCR结果。已经设计了qPCR测定以跨越CSR-1的已知靶转录物的前mRNA的一个内含子。来自三个独立实验(生物学重复)的结果显示为输入的百分比。误差棒代表标准偏差。

笔记

该方案已经应用于表达与Flag表位融合的重组CSR-1蛋白的转基因菌株。然而,它可以与几乎任何在C中表达的Flag标记的核蛋白一起使用。 elegans 。此外,如果针对目标蛋白产生的一级抗体是可获得的,则其可以与蛋白A/G树脂交联并且代替抗Flag M2亲和凝胶使用。在这种情况下,建议选择已在ChIP实验中测试的抗体。由于RNA-ChIP测定的高背景,强烈建议在每个实验中总是使用非转基因菌株作为对照,或者如果使用针对目的蛋白产生的第一抗体,则敲除菌株。以下方案也可以用于细胞质RNA结合蛋白。在这种情况下,可以省略核提取步骤。

食谱

  1. 1x M9缓冲区
    3g KH sub 2 PO 4 sub
    6g Na 2 HPO 4
    17 ml 5 M NaCl
    1ml 1M MgSO 4在1L去离子水中的溶液
  2. 核提取缓冲液
    10mM Tris-HCl(pH7.5) 2mM MgCl 2/
    3mM CaCl 2
    0.5%NP-40
    10%甘油 100 U/ml核糖核酸酶抑制剂
    1x蛋白酶抑制剂鸡尾酒
  3. RIPA缓冲区
    0.1%SDS
    0.1%脱氧胆酸盐 1%Triton X-100 1mM EDTA
    10mM Tris-HCl(pH7.5) 150mM NaCl 100 U/ml RNA酶抑制剂,蛋白酶抑制剂混合物
  4. TSE 150缓冲区
    0.1%SDS
    1%Triton X-100 2mM EDTA 20mM Tris-HCl(pH7.5) 150mM NaCl 100 U/ml核糖核酸酶抑制剂
  5. TSE 500缓冲区
    与TSE 150相同,但使用500 mM NaCl
  6. TSE 1,000缓冲区
    与TSE 150相同,但使用1M NaCl
  7. LiCl缓冲液
    250mM LiCl 1%NP40
    1%脱氧胆酸盐 1mM EDTA
    10mM Tris-HCl(pH7.5) 100 U/ml核糖核酸酶抑制剂
  8. TE缓冲区
    10mM Tris-HCl(pH7.5) 1mM EDTA
  9. 洗脱缓冲液
    10mM Tris-HCl(pH7.5) 0.5%NP-40
    200 mM NaCl
    300μg/ml FLAG肽
    100 U/ml核糖核酸酶抑制剂

致谢

表达与3x FLAG表位融合的重组CSR-1蛋白的转基因菌株由C.Mello(University of Massachusetts,Worcester)友情提供。 这项工作得到美国国家卫生研究院拨款1DP2OD006412-01(A.G。)的支持。 该协议已经从我们以前的工作(Cecere等人,2014年)修改。

参考文献

  1. Bittencourt,D。和Auboeuf,D。(2012)。 通过RNA-ChIP测定分析共转录RNA加工方法 Mol Biol 809:563-577
  2. Cecere,G.,Hoersch,S.,O'Keeffe,S.,Sachidanandam,R.和Grishok,A。(2014)。 CSR-1 RNA干扰途径对转录景观的全球影响 Nat Struct Mol Biol 21(4):358-365。
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cecere, G. and Grishok, A. (2014). RNA Chromatin Immunoprecipitation (RNA-ChIP) in Caenorhabditis elegans. Bio-protocol 4(24): e1358. DOI: 10.21769/BioProtoc.1358.
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