- Open Access
Protocol: a beginner’s guide to the analysis of RNA-directed DNA methylation in plants
© Zhang et al.; licensee BioMed Central Ltd. 2014
- Received: 15 May 2014
- Accepted: 5 June 2014
- Published: 14 June 2014
DNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known.
By using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin.
We have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.
- RNA-directed DNA methylation
- Scaffold RNA
- Pol IV
- Pol V
- Nuclei fractionation
The first part of this procedure is to detect alteration in DNA methylation levels. Alternative to bisulfite sequencing, DNA methylation levels in different samples can be compared by Chop PCR, in which methylation-sensitive enzyme digestion of DNA is followed by PCR amplification of the target sequence (Figure 1B). By using Chop PCR, we developed a method for epigenetic mutant screen that does not need a transgenic reporter gene. A large population of Arabidopsis mutants is commercially available from seed stock sources such as ABRC (Arabidopsis Biological Resource Center) and can be used for Chop PCR-based mutant screen. Genomic DNA samples were prepared by a simple home-made recipe that is suitable for simultaneous DNA extraction from multiple samples. We used the methylation-sensitive restriction enzyme Hae III and examined DNA methylation at AtSN1, a known RdDM target locus. Hundreds of Arabidopsis mutants were screened, resulting in discovery of several DNA hypomethylation mutants including atrrp6l1 alleles that were recently characterized . In addition to Hae III, we have examined some other methylation-sensitive restriction enzymes and correspondingly demonstrated the efficacy of these enzymes in examining several RdDM marker loci in Arabidopsis (Additional file 1: Figure S1; Additional file 2: Table S1). Different restriction enzymes can be used to assay methyaltion in the different sequence contexts, i.e., CG, CHG, and CHH (H represents A, T, or C). Although Chop PCR examines less cytosine than bisulfite sequencing, observation of DNA hypomethylation by Chop PCR in multiple marker loci is sufficient to demonstrate an epigenetic function. Quantification of the difference in DNA methylation levels can be achieved by Chop qPCR (Additional file 1: Figure S1B). Parallel amplification of the marker locus from non-digested DNA samples is commonly used to show loading controls. However, we recommend amplifying DNA sequences without restriction sites, from the same digested DNA samples, as internal controls, because experimental accuracy can be increased while the amount of required DNA can be reduced. Compared to traditional forward genetic screens that involve transgenic reporter genes, Chop PCR-based screen does not require generation of a transgenic reporter line and the subsequent mutated population, neither required is the process of map-based cloning that can be time-consuming.
The second part of this procedure is quantification of siRNAs (Figure 1C). The levels of siRNAs can be semi-quantitatively detected by RNA hybridization, which typically uses ≥20 μg RNAs that are enriched in sizes smaller than 200 bp . The whole process takes several days from RNA extraction to final results. Alternative to RNA hybridization, small RNAs can be detected by RT-qPCR that employs stem loop primers during reverse transcription . This approach takes only a few hours starting from RNA extraction and involves the patented TaqMan Small RNA Assays. However, the protocol of TaqMan Small RNA Assays, which was initially implemented to detect miRNAs, does not readily enable detection of lower abundance small RNAs such as 24 nt siRNAs that mediate RdDM. In addition, these PCR-based quantitative measurements require, as a prerequisite, knowing the exact sequences of small RNAs; meanwhile mining published small RNA datasets for such information typically requires skills in bioinformatics. Therefore we developed a simple bioinformatics method for selecting siRNAs to quantify and optimized the TaqMan Small RNA Assays protocol for detecting 24 nt siRNAs.
To obtain nucleotide sequences for 24 nt siRNAs from the regions of interest, we searched small RNA sequencing databases. This can be done by BLAST searches of published small RNA databases, such as Zhang et al. , for siRNAs that match the regions of interest. However, such analysis requires skills in bioinformatics. We thus provide an alternative analysis approach that can be applied in any laboratory, by using Arabidopsis AtSN1 locus as an example. AtSN1 locates within 1 kb downstream of the gene At3g44006 . Browsing At3g44006 and its downstream region in IGB (Integrated Genome Brower) identified AtSN1 as At3TE63860, which is a transposable element that starts at 15794808 and ends at 15794619. Arabidopsis small RNA nucleotide sequence information with genomic coordinates is available in the UDelaware Small RNA database . Since 24 nt siRNAs are proposed to pair with scaffold RNAs [1–4], we screened AtSN1 small RNAs for 24 nt siRNAs that are annotated as Waston strand, i.e., template DNA strand. This means that these siRNAs have the same sequences as the template DNA strand (despite the U/T difference) and thus can potentially pair with scaffold RNAs. To increase the chance of detecting individual siRNAs, these 24 nt siRNAs were subjected to a second screen for those that showed abundance of ≥ 1 hit per million reads, according to the UDelaware Small RNA database, in the wild type Col-0 ecotype. Among these candidates, one siRNA exhibited sequence homology to a 43 nt probe that had been used for detecting AtSN1 siRNAs by Northern blots . Thus we chose this siRNA for customer TaqMan Small RNA Assay design.
The effectiveness of using TaqMan Assays to quantify 24nt siRNA levels was confirmed by using the Arabidopsis mutants nrpd1-3 that is defective in RNA polymerase Pol IV, which is required for biogenesis of almost all 24nt siRNAs , and nrpe1-11 that is defective in Pol V, of which mutation decreases the levels of some Pol IV-dependent siRNAs . Compared to wild type plants (Col-0), nrpd1-3 exhibited depletion of all examined siRNAs including AtSN1, siR1003, solo LTR, 5TE27040, IGN5B, and Cluster 2 (Figure 2A). The nrpe1-11 mutant also showed substantial reduction in the levels of the examined siRNAs except Cluster 2 (Figure 2A), which is known to be Pol IV-dependent and Pol V-independent . The patterns observed in our RT-qPCR measurements are consistent with published RNA hybridization results [10, 17] and whole genome small RNA sequencing results . Sequences of these individual 24nt siRNAs are listed in Additional file 2: Table S2. Designing siRNA probes requires only submitting individual siRNA sequences to the manufacturer website of the patented TaqMan Assays.
The third part of studying RdDM nucleic acids is to quantitatively measure chromatin-associated scaffold RNAs. In Arabidopsis, Pol V transcription generates scaffold RNAs that are of low abundance and are devoid of poly-A tail . Therefore we used scaffold RNA-specific primers during reverse transcription for the first-strand cDNA synthesis, and subsequently performed nested qPCR. Such an approach successfully measures Pol V-transcribed scaffold RNAs, as demonstrated by comparison between Col-0 and nrpe1-11 (Figure 2B). Although the levels of scaffold RNAs are in proportion to the pool of total Pol V transcripts, sometimes nuclei fractionation is needed to distinguish chromatin-associated scaffold RNAs from those Pol V transcripts that have been released from chromatin (Figure 1D), as was the case in our recent study of AtRRP6L1 that functions in retention of Pol V transcripts at chromatin . Nuclei can be isolated with Honda buffer and subsequently lysed in the presence of urea and detergent, followed by centrifugation that sediments chromatin-associated RNAs in the pellet (chromatin-associated fraction), while leaving released RNAs in the supernatant (chromatin-free fraction) (Figure 1D). As an indication of successful nuclei fractionation, genomic DNA should be nearly absent in the chromatin-free fractions (Additional file 1: Figure S3).
To provide an easy-to-follow procedure, experimental details are described step by step in the Methods.
To summarize, we have developed an integrative procedure for studying nucleic acids that are central players in RNA-directed DNA methylation and other epigenetic regulation (Figure 1). The results not only demonstrated the efficacy of this procedure (Figure 2; Additional file 1: Figure S1-S3), but also provide a list of novel RdDM marker loci (Additional file 2: Table S1) and the related methylation-sensitive restriction enzymes (Additional file 2: Table S1), as well as the nucleotide sequences of a list of individual 24nt siRNA (Additional file 2: Table S2), which can be valuable for future epigenetic studies. Moreover, this procedure requires little previous experimental experience in epigenetics or bioinformatics, and thus can broadly benefit the scientific community.
<I-1> Preparation of genomic DNA samples
Prepare 1.5 mL eppendorf tubes, each containing 3 Chrome steel beads (BioSpec Products, Inc, Cat.# 11079132C).
* Note: Steps 1 to 3 are optimized for preparing multiple samples. Start from Step 4 if samples are homogenized by other methods.
Take one or two mature leaves and put into labeled tubes. Make sure the tubes are tightly capped.
Freeze tubes in liquid nitrogen shortly, then shake tubes vigorously in a box to shatter the samples.
*Note: Be aware that a capped tube with liquid nitrogen in it will pop open or even explode under room temperature.
Add 500 μL 2% CTAB (Cetyltrimethyl ammonium bromide) solution to each tube (or per ~100 mg sample if homogenized by other methods), then incubate at 65°C for 30-60 min.
* Note: To make 100 mL CTAB solution (modified from ): 2g CTAB, 10 mL 1 M Tris-HCl (pH = 7.5), 28 mL 5 M NaCl, 4 mL 0.5 M EDTA (pH = 8.0), H2O.
Add 500 μL chloroform, then votex vigorously and centrifuge at ≥ 12000 g at room temperature for 10 min.
Transfer aqueous phase into new tubes, then mix with equal volume isopropanol.
Store at -20°C for ≥ 30 min.
Centrifuge at ≥ 12000 g at room temperature for 10 min.
Wash the pellet with 0.5 mL 70% ethanol.
Air dry pellets and dissolve in 50 μL nuclease-free water (we use autoclaved double-distilled water).
Proceed to steps in < I-2 > for Chop-PCR if doing mutant screening. Alternatively, for Chop-qPCR, continue with DNA purification as described below.
* Note: Although DNA samples extracted by this way have RNA contamination, they can be used for rough estimations of DNA methylation levels during initial mutant screen. RNase treatments should be performed for Chop qPCR and for semi-quantitative Chop PCR that is used for the purposes other than rough estimation.
Add 2 μL RNase A (10mg/mL) per 500 μL DNA solution, mix well and incubate at 37°C for 1 hr.
* Note 1: Concentration of DNA solutions can vary depending on the yield of crude extraction using CTAB solution.
* Note 2: If making RNase A solution from powders, boiling the solution for 15–20 minutes usually eliminate most of its DNases.
Add equal volume phenol: chloroform: isoamyl alcohol (24:1:1, pH 8.0), vortex vigorously for 5 sec, then centrifuge at ≥ 12000 g at room temperature for 10 min.
(Optional) Transfer the aqueous phase to a new tube, repeat Step 2.
Transfer the aqueous phase to a new tube, add equal volume phenol: chloroform, vortex vigorously for 5 sec, then centrifuge at ≥ 12000 g at room temperature for 8 min.
Transfer the aqueous phase to a new tube, add 2 volume ethanol, 0.1 volume NaOAc (pH 5.2, 3 M), mix well.
Store at -20°C for ≥ 30 min.
Centrifuge at ≥ 12000g at room temperature for 10 min.
Wash the pellet 2X with 0.5 mL 70% ethanol.
Air dry pellets and dissolve in nuclease-free water.
<I-2 > Enzymatic DNA digestion and PCR/qPCR
Measure genomic DNA concentration using Nanodrop.
Digest equal amount (typically 0.5 to 1 μg) of genomic DNA with a methylation-sensitive enzyme in a 20 μl reaction mixture.
* Note: While following manufacture protocols to set up reactions for each enzyme, the digestion time can be extended to overnight.
After digestion, check DNA methylation levels at the enzyme-targeted marker locus by qPCR or PCR. For semi-quantitative PCR,use 1 μL of the digested DNA as template in a 20 μl reaction mixture that contains 0.15 unit of commercial Taq DNA polymerase. Run a DNA gel (1.2% agarose) to compare the PCR results from different samples. For qPCR, prepare 20 μl reaction mixture following manufacture protocols and use 1 μl of the digested DNA as template per reaction.
* Note 1: DNA input amounts in different samples should be normalized by amplifying an internal control sequence that does not contain the restriction site of the enzyme used. Alternatively, non-digested DNA can be used as PCR/qPCR templates for input normalization.
* Note 2: In semi-quantitative PCR, too many PCR cycles will probably yield in saturated amplification and thereby mask small differences in template inputs. For optimized observation of differences, PCR can be repeated with increased or decreased cycle numbers after initial assessment. A positive control, e.g., the Pol IV mutant nrpd1-3, should be examined in parallel to the wild type.
Optional: For Chop-qPCR, follow steps 4 to 10 to analyze the results
Export qPCR results to Excel file.
Calculate average Ct value for the non-digested control in each sample.
Calculate “ΔCt = Ct of the examined amplicons – average Ct of the non-digested control”.
Calculate average ΔCt value of one sample (e.g., wild type plants).
Calculate the comparative Ct values (ΔΔCt) for other samples (e.g., mutant plants). ΔΔCt = ΔCt (mutant) – average ΔCt (wild type).
Calculate relative abundance of the amplicons by using this function: “= 2^(-ΔΔCt)”.
Calculate average of relative abundance of the amplicons; and calculate standard deviation by using the function “=STDEV(values to be calculated)”.
II. siRNA RT-qPCR
<II-1> Isolation of small RNAs
Homogenize ≤100 mg plant tissues in liquid nitrogen by using mortar and pestle. Transfer the powder into a 1.5 mL eppendorf tube (pre-chilled in liquid nitrogen) and add 1 mL RNAzol® RT (Molecular Research Center Inc)
Add 0.4 mL water per 1 mL of RNAzol® RT. Mix well and store at room temperature for 5-15 min.
* Note: Autoclaved distilled water is good enough for this step.
Centrifuge the mixture ≥12,000 g at room temperature for 15 min.
* Note: this step precipitates DNA, protein, and polysaccharide.
Transfer the supernatant to a new tube and add 0.4 mL 75% ethanol per 1 mL of supernatant. Mix well and store at room temperature for 10 min.
Centrifuge ≥12,000 g for 8 min at room temperature or at 4°C.
* Note: this step pellets RNA > 200 bp.
Transfer the supernatant to a new tube and add 0.8 volume of isopropanol. Mix well and store at 4°C for 30 min.
* Note: the mixture can be stored at -20°C for longer time.
Centrifuge ≥12,000g for 15 min at room temperature or at 4°C.
Wash the pellet with 70% isopropanol twice.
* Note: After removing the bulk isopropanol solution, centrifuge briefly and use pipette to remove the residual liquid. By doing this, RNA pellets dissolved without drying perform well in the downstream RT-PCR reactions.
Dissolve the pellet in 20-40 μL RNase-free water, without drying, in RNase-free water. Keep on ice and proceed to reverse transcription, or store at -80°C.
<II-2> Quantitative measurement of siRNAs by RT-qPCR
Search small RNA databases to determine sequences of siRNAs to be examined (see main text for an example).
* Note: Additional file 2: Table S2 lists some Arabidopsis 24nt siRNAs that have been detected by RT-qPCR in this study.
Submit siRNA sequences to Life Technologies for TaqMan Small RNA Assay design by following the online instructions.
Place the order after the designing has been completed.
* Note: we suggest downloading the TaqMan Small RNA Assay protocol at this point, as well as to order the TaqMan MicroRNA Reverse Transcription Kit and the TaqMan Universal PCR Master Mix as recommended in the protocol.
- 4.Use TaqMan MicroRNA Reverse Transcription Kit to prepare reverse transcription (RT) master mixture and keep on ice. The following recipe is for one reaction:
100 mM dNTPs 0.15 μL
MultiScribe TM Reverse Transcriptase (50 unit/μL) 1.0 μL
10X Reverse Transcription Buffer 1.5 μL
RNaseOut (40 unit/μL) 0.9 μL
RNase-free Water 4.26 μL
Total Volume: 7 μL
In an RNase-free PCR tube, add 400 ng small RNA in a volume of 5 μL. Then add 2 μL snoR101 RT primer and 2 μL siRNA RT primer. Mix well gently.
400ng small RNA is sufficient for consistently detecting all 24 nt siRNAs that we have examined. For certain abundant siRNAs such as siR1003 that originates from 5S rDNA repeats, 100 ng small RNA is sufficient.
Incubate the RNA-primers mixture at 85°C for 5 min, followed by 60°C for 5 min. Then put the mixture on ice immediately and for at least 1 min.
Add 7 μL RT mixture to each RNA-primers mixture. Mix well gently. Keep in ice for 5 min.
Perform RT reaction using the following conditions consecutively: 16°C for 30 min, 42°C for 30 min, 85°C for 5 min. Keep the samples on ice after RT reaction.* Note: Because TaqMan stem-loop primer recognizes a specific short sequence and the qPCR reverse primer is complementary to the stem-loop RT primers (see Figure 1C in main text), genomic DNA contamination, if any, will not be a problem for this experiment. Thus no-RT control is unnecessary. In fact, we did not detect qPCR signals even using genomic DNA.
- 9.Prepare the qPCR mixture as the following:
TaqMan Small RNA Assay (20×) 1.0 μL
cDNA from RT reaction (Step 8) 1.2 μL
TaqMan Universal PCR Master Mix II (2×) 10.0 μL
Nuclease-free water 7.8 μL
Total Volume: 20 μL
* Note 1: run qPCR individually for snoR101 and the siRNA and perform 3 technical repeats.
* Note 2: The patented TaqMan Small RNA Assays provide qPCR primers in the tubes labeled as “TaqMan Small RNA Assay (20×)” without giving primers information.
- 10.Perform qPCR in a real-time PCR instrument using the following program:
Step I: 50°C (optional) 2 min
Step II: 95°C (Enzyme activation) 10 min
Step III: 95°C (Template denaturing) 15 sec
Step IV: 60°C (Anneal/extend) 1 min
Step V: go to Step III, 39 times (totally 40 cycles)
Step VI: End
* Note: TaqMan Small RNA Assay PCR signals will be detected in the FAM channel by the qPCR instrument.
Export results to Excel file.
Calculate average Ct value for snoR101 in different replicates.
Calculate “ΔCt = Ct of siRNA – average Ct of snoR101” from the same RT reaction.
Calculate average ΔCt value of one RNA sample (e.g., wild type plants).
Calculate the comparative Ct values (ΔΔCt) for other samples (e.g., mutant plants). ΔΔCt = ΔCt (mutant) – average ΔCt (wild type).
Calculate relative siRNA abundance by using this function: “= 2^(-ΔΔCt)”.
Calculate average of relative siRNA abundance; and calculate standard deviation by using the function “=STDEV(values to be calculated)”.
* Note: Mean value and standard deviation can also be calculated based on multiple biological replicates. The expression patterns, e.g., release of transcriptional silencing, should always be the same for different biological replicates, although the standard deviation between different biological replicates is usually greater than that of technical repeats from one single biological replicate.
III. Scaffold RNA RT-qPCR
<III-1> Quantitative measurement of scaffold RNAs without nuclei fractionation
Extract total RNA by using Trizol (Invitrogen) following the manufacturer’s protocol.
* Note: Scaffold RNAs may be a few hundred nucleotides, so for this experiment we do not recommend using those commercial RNA extraction kits that preferentially extract RNA > 200 bp.
Remove DNA contamination in the RNA samples by using TURBO DNA-free™ (Ambion).
* Note: We typically treat 10 μg RNA with TURBO DNase in a 20 μL reaction, and use maximum amount of RNA during RT reaction while following the manufacturer RT protocol.
Perform RT reaction by using SuperScript™ III First-Strand Synthesis SuperMix (Invitrogen). In each reaction, use mixed gene/scaffold RNA-specific primers (the scaffold RNA and the internal control ACTIN2). For a 10 μL reaction, use 0.5 μL pre-mixed primers (2 mM each).
* Note 1: We have tried using some other RT kits and found that, in our hands, SuperScript works best for low-abundance non-coding RNA detection.
* Note 2: We recommend using the same amount of RNA for each RT reaction. We typically use 1 μg total RNA for a reaction in 10 μL.
* Note 3: No-RT control reactions can be performed by making RT mixtures as describe above but without reverse transcriptase. This will ensure no false qPCR signals from DNA contamination in RNA samples.
- 4.Perform qPCR by using SYBR® Green Master Mix. PCR for scaffold RNA and ACTIN2 should be separated.
Step I: 95°C 3 min
Step II: 95°C 10 sec
Step III: Tm 30 sec
Step IV: go to Step II 39 times + plate read (totally 40 cycles)
Step V: increase from 65°C to 95°C, increment 0.5°C for 5 sec, plus plate read
Step VI: End
* Note 1: Because cDNAs are synthesized by using gene-/scaffold RNA-specific primers, PCR amplification that follows has to be nested PCR, i.e., positions of the qPCR primers on the DNA coding strand should be upstream of the gene-/scaffold RNA-specific RT primers.
* Note 2: A single peak in the melt curve generated by Step V should indicate the specificity of the primers. This can also be examined by DNA gel electrophoresis.
Use ACTIN2 as internal control to analyze the results (Refer to Steps V-11 to V-17 for details).
* Note: By starting with equal amount of RNA, different samples should show similar Ct value for ACTIN2 in qPCR reactions. Alternatively, another housekeeping gene can be examined to ensure similar RNA quality.
<III-2> Quantitative measurement of scaffold RNAs with nuclei fractionation
Prepare Honda buffer (0.4 M Sucrose, 2.5% Ficoll, 5% Dextran T40, 25 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 0.5% Triton X-100, 0.5 mM PMSF, 0.1% pepstatin A, 0.1% aprotinin, 10 mM β-mercaptoethanol, 8 unit/mL RNaseOUT) (modified from ).
* Note: add PMSF, pepstatin A, aprotinin, β-mercaptoethanol, and RNaseOUT before use.
Grind 3 gram ~12-day-old plants with liquid nitrogen.
* Note: We recommend using the same amount of plant materials for each sample.
Transfer the power to a 50 mL Falcon tube prefilled with 15 mL cold Honda buffer. Homogenize by vortex vigorously. Keep the tubes in ice for 15-30 min.
Filter the homogenate through 4 layers of Miracloth, followed by centrifugation at 1500 g at 4°C for 5 min.
Re-suspend the pellet in 1 mL Honda buffer, then transfer to a new 1.5 mL eppendorf tube, followed by centrifugation at 1800 g at 4°C for 5 min to pellet the nuclei.
* Note: Save the supernatant for cytosol analysis if wished.
Gently rinse the nuclei pellet with ice-cold wash buffer (1X PBS, 1 mM EDTA, 8 unit/mL RNaseOUT).
Re-suspend the pellet in 0.5 mL prechilled glycerol buffer (20 mM Tris-HCl, pH 7.9, 75 mM NaCl, 0.5 mM EDTA, 0.85 mM DTT, 50% glycerol, 0.125 mM PMSF, 0.1% pepstatin A, 0.1% aprotinin, 10 mM β-mercaptoethanol, 160 unit/mL RNaseOUT) by gently flicking the tube.
Add 0.5 mL cold nuclei lysis buffer (10 mM HEPES, pH 7.6, 1mM DTT, 7.5 mM MgCl2, 0.2 mM EDTA, 0.3 M NaCl, 1 M Urea, 1% NP-40, 0.5 mM PMSF, Proteinase inhibitor cocktail, 0.1% pepstatin A, 0.1% aprotinin, 10 mM β-mercaptoethanol, 160 unit/mL RNaseOUT).
Gently vortex for 2 × 2 sec, incubate on ice for 2 min, centrifuge at 14,000 rpm at 4°C for 2 min to separate the chromatin fraction and the nucleoplasmic fraction.
Treat the supernatant (nucleoplasmic fraction) with proteinase K at 37°C for 1 hr, followed by traditional acidic phenol/chloroform extraction and ethanol precipitation for RNA.
Gently rinse the chromatin pellet twice with 0.5 mL cold wash buffer and then dissolve in 1 mL Trizol for RNA extraction.
Refer to Section III-1 for RT-qPCR of scaffold RNAs.
This work was supported by US National Institutes of Health grants to J-K Zhu. We thank Rebecca Stevenson and the reviewers for critical reading of the manuscript.
- Matzke M, Kanno T, Daxinger L, Huettel B, Matzke AJM: RNA-mediated chromatin-based silencing in plants. Curr Opin Cell Biol. 2009, 21: 367-376. 10.1016/j.ceb.2009.01.025.View ArticlePubMedGoogle Scholar
- Law JA, Jacobsen SE: Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet. 2010, 11: 204-220. 10.1038/nrg2719.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang H, Zhu JK: RNA-directed DNA methylation. Curr Opin Plant Biol. 2011, 14: 142-147. 10.1016/j.pbi.2011.02.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Pikaard CS, Haag JR, Pontes OMF, Blevins T, Cocklin R: A transcription fork model for Pol IV and Pol V-dependent RNA-directed DNA methylation. Cold Spring Harb Symp Quant Biol. 2012, 77: 205-212. 10.1101/sqb.2013.77.014803.View ArticlePubMedGoogle Scholar
- Watanabe T, Tomizawa SI, Mitsuya K, Totoki Y, Yamamoto Y, Kuramochi-Miyagawa S, Iida N, Hoki Y, Murphy PJ, Toyoda A, Gotoh K, Hiura H, Arima T, Fujiyama A, Sado T, Shibata T, Nakano T, Lin H, Ichiyanagi K, Soloway PD, Sasaki H: Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus. Science. 2011, 332: 848-852. 10.1126/science.1203919.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang H, Zhu JK: Active DNA demethylation in plants and animals. Cold Spring Harb Symp Quant Biol. 2012, 77: 161-173. 10.1101/sqb.2012.77.014936.PubMed CentralView ArticlePubMedGoogle Scholar
- Saleh A, Alvarez-Venegas RUL, Avramova Z: An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in Arabidopsis plants. Nat Protoc. 2008, 3: 1018-1025. 10.1038/nprot.2008.66.View ArticlePubMedGoogle Scholar
- Kasinathan S, Orsi GA, Zentner GE, Ahmad K, Henikoff S: High-resolution mapping of transcription factor binding sites on native chromatin. Nat Methods. 2014, 11: 203-209.PubMed CentralView ArticlePubMedGoogle Scholar
- Wierzbicki AT, Haag JR, Pikaard CS: Noncoding transcription by RNA polymerase Pol IVb/Pol V mediates transcriptional silencing of overlapping and adjacent genes. Cell. 2008, 135: 635-648. 10.1016/j.cell.2008.09.035.PubMed CentralView ArticlePubMedGoogle Scholar
- He XJ, Hsu YF, Pontes O, Zhu J, Lu J, Bressan RA, Pikaard C, Wang CS, Zhu JK: NRPD4, a protein related to the RPB4 subunit of RNA polymerase II, is a component of RNA polymerases IV and V and is required for RNA-directed DNA methylation. Genes Dev. 2009, 23: 318-330. 10.1101/gad.1765209.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang H, Tang K, Qian W, Duan C, Wang B, Zhang H, Wang P, Zhu X, Lang Z, Yang Y, Zhu JK: An Rrp6-like protein positively regulates non-coding RNA levels and DNA methylation in Arabidopsis. Mol Cell. in pressGoogle Scholar
- Xie Z, Johansen LK, Gustafson AM, Kasschau KD, Lellis AD, Zilberman D, Jacobsen SE, Carrington JC: Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2004, 2: E104-10.1371/journal.pbio.0020104.PubMed CentralView ArticlePubMedGoogle Scholar
- Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ: Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005, 33: e179-e179. 10.1093/nar/gni178.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang X, Henderson IR, Lu C, Green PJ, Jacobsen SE: Role of RNA polymerase IV in plant small RNA metabolism. Proc Natl Acad Sci U S A. 2007, 104: 4536-4541. 10.1073/pnas.0611456104.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakano M, Nobuta K, Vemaraju K, Tej SS, Skogen JW, Meyers BC: Plant MPSS databases: signature-based transcriptional resources for analyses of mRNA and small RNA. Nucleic Acids Res. 2006, 34 (Database issue): D731-5.PubMed CentralView ArticlePubMedGoogle Scholar
- Mosher RA, Schwach F, Studholme D, Baulcombe DC: PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis. Proc Natl Acad Sci U S A. 2008, 105: 3145-3150. 10.1073/pnas.0709632105.PubMed CentralView ArticlePubMedGoogle Scholar
- Zheng B, Wang Z, Li S, Yu B, Liu JY, Chen X: Intergenic transcription by RNA polymerase II coordinates Pol IV and Pol V in siRNA-directed transcriptional gene silencing in Arabidopsis. Genes Dev. 2009, 23: 2850-2860. 10.1101/gad.1868009.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang H, Ma ZY, Zeng L, Tanaka K, Zhang CJ, Ma J, Bai G, Wang P, Zhang SW, Liu ZW, Cai T, Tang K, Liu R, Shi X, He XJ, Zhu JK: DTF1 is a core component of RNA-directed DNA methylation and may assist in the recruitment of Pol IV. Proc Natl Acad Sci U S A. 2013, 110: 8290-8295. 10.1073/pnas.1300585110.PubMed CentralView ArticlePubMedGoogle Scholar
- Clarke JD: Cetyltrimethyl Ammonium Bromide (CTAB) DNA miniprep for plant DNA isolation. Cold Spring Harb Protoc. 2009, doi: 10.1101/pdb.prot5177Google Scholar
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