This work was supported by the National Natural Science Foundation of China (grant no. 31600250, Y.Z.), Science and Technology Projects of Guangzhou City (grant no. 201804020015, H.N.), and the China Agricultural Research System (grant no. CARS-04-PS09, H.N.).

1. Primer Design
<ol>
	<li>Design gene-specific primers for reverse transcription (RT) using PCR primer design software (Table of Materials) based on the general rules of primer design17. 
	NOTE: RT primers are highly specific to the target transcripts, and they are generally anchored on the 5&#8217; part of coding sequences (mature mRNAs and precursor RNAs), or ~500&#8211;600 nt downstream of the anticipated 5&#8217; end (18S and 26S rRNAs).</li>
	<li>Design pairs of divergent primers to amplify the circularized transcripts by cRT-PCR. 
	NOTE: The paired divergent primers flank the 5&#8217;-3&#8217; junction of the circularized transcripts, and their positions vary among the target transcripts analyzed (Figure S1). Some transcripts have long UTRs; for example, nad2-1 5&#8217; UTR and rps4-1 3&#8217; UTR are 1,985 and 1,826/1,834 nt, respectively1. If both primers are anchored on coding region, it will be hard to amplify the target transcripts. By contrast, some UTRs are short; for example, the 3&#8217; UTRs of nad2-1 and nad4-1 are 34/35 and 29&#8211;31 nt, respectively1. If the paired primers are located far away from the coding sequences, the PCR will fail. Generally, two pairs of divergent primers are designed for each target transcripts, while multiple pairs may be necessary to map those whose transcript patterns are complicated and/or UTRs are very long.</li>
	<li>Design pairs of convergent primers to prepare RNA probes for Northern blot. 
	NOTE: The paired primers are located on coding region of the target gene, and the size of the PCR product should be in the range of 100 to 1,000 bp. Each forward primer should contain a restriction enzyme site to minimize vector sequences in the resulting probes.</li>
</ol>
2. Preparation of Crude Mitochondrion from Maize Developing Kernels
<ol>
	<li>Sterilize pestles, mortars, glass funnels, tubes, and tips by autoclave, and dry them in oven.</li>
	<li>Perform all procedures at 4 &#176;C or on ice, and pre-cool all solutions.</li>
	<li>Prepare 100 mL of extraction buffer (EB), composed of 0.3 M sucrose, 5 mM tetrasodium pyrophosphate, 10 mM KH2PO4, 2 mM EDTA, 1% [w/v] polyvinylpyrrolidone 40, 1% [w/v] bovine serum albumin, 5 mM L-cysteine, and 20 mM ascorbic acid. Adjust to pH 7.3 with KOH and sterilize by filtration.</li>
	<li>Prepare 100 mL of wash buffer (WB) consisting of 0.3 M sucrose, 1 mM EGTA, and 10 mM MOPS (3-(N-morpholino)propanesulfonic acid). Adjust to pH 7.2 with NaOH and sterilize by filtration. 
	NOTE: It is suggested to prepare EB and WB using DEPC-treated deionized H2O.</li>
	<li>Collect 20 g developing kernels at 11&#8211;20 days after pollination (DAP) to a 50-mL tube placed on ice, and then transfer the kernels to pre-cooled mortars. 
	NOTE: Use a ratio of 100 mL of EB to 20 g of maize kernels.</li>
	<li>Add 10&#8211;20 mL of ice-cold EB to each mortar, and grind the kernels completely.</li>
	<li>Add more EB, and filter the ground tissues through two layers of filter cloth (Table of Materials).</li>
	<li>Centrifuge the filtrate at 8,000 x g for 10 min, and discard the pellet.</li>
	<li>Transfer the supernatant to a new tube, and centrifuge it at 20,000 x g for 10 min.</li>
	<li>Pour off the supernatant, and resuspend the pellet in 6 mL WB.</li>
	<li>Aliquot the suspension to five 1.5 mL RNase-free tubes, and centrifuge them at 14,000 x g for 5 min.</li>
	<li>Discard the supernatant, freeze the mitochondrial pellet in liquid nitrogen, and store at -80 &#176;C.</li>
</ol>
3. Extraction of Mitochondrial RNA
<ol>
	<li>Extract mitochondrial RNA with a commercial reagent (Table of Materials) according to the manufacture&#8217;s instructions. 
	CAUTION: This reagent contains phenol and guanidine isothiocyanate. Work with it in a fume hood, and wear lab coat and gloves.</li>
	<li>Dissolve the isolated mitochondrial RNA in DEPC-treated deionized H2O, and estimate RNA concentration and purity with a spectrophotometer (Table of Materials). 
	NOTE: Generally, ~250 &#956;g mitochondrial RNA is obtained from 20 g of 15-DAP maize kernels.</li>
	<li>Prepare one agarose gel composed of 1x TAE buffer (40 mM Tris, 20 mM acetic acid, 1mM EDTA), 1.5% agarose (Table of Materials), and 1x nuclear staining dye (Table of Materials).</li>
	<li>Add an appropriate volume of 10x loading buffer (0.5% bromophenol blue, 0.5% xylene cyanol FF, and 50% glycerol), and load mitochondrial RNA/Loading buffer mixture on the 1.5% agarose gel.</li>
	<li>Run the gel in 1x TAE buffer at 5&#8211;6 V/cm for 20&#8211;25 min, and evaluate mitochondrial RNA integrity by imaging the gel with a gel documentation system (Table of Materials). 
	NOTE: The presence of two distinct bands (~3,510 and ~1,970 nt for maize mitochondrial 26S and 18S rRNAs, respectively) is an acceptable standard for intact mitochondrial RNA. To exclude possible degradation, RNA integrity should be investigated during the multiple steps of circularized RNA preparation (Figure 2).</li>
</ol>
4. RNA 5&#8217; Polyphosphatase Treatment
<ol>
	<li>Set up RNA 5&#8217; polyphosphatase (Table and Materials) treatment (Table 1), and incubate at 37 &#176;C for 30&#8211;60 min.</li>
	<li>Recover the 5&#8217; polyphosphatase-treated RNA with an RNA purification kit (Table of Materials) according to the manufacturer&#8217;s instructions.</li>
	<li>Repeat steps 3.3 to 3.5.</li>
</ol>
5. RNA Circularization
<ol>
	<li>Prepare two circularization reactions using the same amounts of 5&#8217; polyphosphatase-treated and non-treated mitochondrial RNAs (Table 2), and incubate both reactions at 16 &#176;C for 12&#8211;16 h.</li>
	<li>Recover the two sets of self-ligated RNAs using the same kit as in step 4.2. 
	NOTE: It should be noted that only a fraction of mitochondrial RNA will be self-ligated, and the recovered RNA will be a mixture of linear and circularized transcripts.</li>
	<li>Repeat steps 3.3 to 3.5.</li>
</ol>
6. Reverse Transcription
<ol>
	<li>Synthesize two sets of cDNAs from the same amounts of circularized 5&#8217; polyphosphatase-treated and non-treated RNAs (200 ng).</li>
	<li>Prepare a primer mixture by adding an equal ratio of 26S-CRT and up to 7 other RT primers. 
	NOTE: The final concentration of the primer mixture should be 1 &#956;M.</li>
	<li>Prepare two pre-mixtures by combining the reagents listed in Table 3, incubate at 65 &#176;C for 5 min, and then chill on ice for 2 min.</li>
	<li>Assemble two RT reaction systems (Table 4), and incubate them at 42 &#176;C for 50 min.</li>
	<li>Heat both RT reactions at 70 &#176;C for 5 min, and then chill them on ice.</li>
</ol>
7. Normalization
<ol>
	<li>Prepare two PCR reactions by adding the same volume of template cDNAs derived from 5&#8217; polyphosphatase-treated or non-treated RNAs, divergent primers flanking the 5&#8217;-3&#8217; junction of circularized 26S rRNA (i.e. 26S-CF1 and -CR1), etc. (Table 5).</li>
	<li>Run the two reactions in a thermal cycler (Table of Materials) under conditions described in Table 6.</li>
	<li>Prepare one agarose gel composed of 1x TAE buffer, 1.0% agarose, and 1x nuclear staining dye.</li>
	<li>Add 2 &#956;L 10x loading buffer (0.5% bromophenol blue, 0.5% xylene cyanol FF, and 50% glycerol) to each of the two PCR products, and load them on the gel.</li>
	<li>Run the gel in 1x TAE buffer at 5&#8211;6 V/cm for 30&#8211;40 min, and image it using the same gel documentation system as step 3.5.</li>
	<li>Compare the abundance of the two PCR products using computer software (Table of Materials, Figure 4A), and optimize the normalization by changing the amounts of template cDNAs if necessary.</li>
</ol>
8. PCR Amplification
<ol>
	<li>Prepare pairs of PCR reactions by adding appropriate volume of the normalized cDNAs and a pair of divergent primers flanking 5&#8217;-3&#8217; junction of the target transcripts (Table 7). 
	NOTE: The amount of template cDNAs used for PCR amplification of the target transcripts are determined by the 26S rRNA normalization results.</li>
	<li>Perform the PCR reactions according to the program described in Table 8.</li>
	<li>Repeat steps 7.3 to 7.5.</li>
	<li>Change to nested divergent primers, and verify the first round PCR results by repeating steps 8.1 and 8.2.</li>
	<li>Repeat steps 7.3 to 7.5.</li>
	<li>Recover the prominent bands that could be repeated in two rounds of PCR amplification using a gel DNA recovery kit (Table of Materials).</li>
</ol>
9. Determination of Transcript Termini
<ol>
	<li>Clone the gel-purified PCR products into a blunt-end vector (Table of Materials) using standard techniques.</li>
	<li>Perform colony PCR to select positive clones containing the target inserts, and sequence them commercially. 
	NOTE: Positive clones containing inserts with variable size are usually detected from a single recovered band because many steady-state transcripts in plant mitochondrial have heterogeneous 5&#8217; and/or 3&#8217; ends1,12.</li>
	<li>Align the sequencing data with maize mitochondrial genome using basic local alignment search tool (BLAST) of national center for biotechnology information (NCBI). Choose organism &#8220;maize (taxid:4577)&#8221;, and search database &#8220;Nucleotide collection (nr/nt)&#8221;.</li>
	<li>Find the 5&#8217;-3&#8217; junction of the circularized transcript, and determine the positions of 5&#8217; and 3&#8217; transcript termini.</li>
	<li>Calculate the size of target transcripts.</li>
</ol>
10. Verification of the cRT-PCR Mapping Results by RNA Gel Blot Hybridization
NOTE: RNA gel blot hybridization is performed by using a commercial kit (Table of Materials), which contains the reagents for transcription-labeling of RNA with digoxigenin (DIG) and T7 RNA polymerase, hybridization, and immunological detection. Please refer to the protocols provided in this kit for more details. Make sure that only RNase free equipment is used for the whole procedure.
<ol>
	<li>Amplify the DNA fragment used to prepare RNA probe, and clone it to the same vector as in step 9.1, which contains a T7 promoter 17 bp upstream of the insertion site.</li>
	<li>Linearize the construct using proper restriction enzyme, recover the linearized plasmid by a DNA purification kit (Table of Materials), and dissolve it in DEPC-treated deionized H2O.</li>
	<li>Label RNA probes with DIG-11-UTP by a commercial kit (Table of Materials) according to the manufacturer&#8217;s instructions.</li>
	<li>Prepare 500 mL of 10x MOPS buffer (0.2 M MOPS, 50 mM sodium acetate, and 10 mM EDTA) using DEPC-treated deionized H2O. Adjust to pH 7.0 by NaOH, and sterilize by filtration.</li>
	<li>Add 2&#8211;3 volumes of loading buffer (50% formamide, 6.2% formaldehyde, 1x MOPS, 10% glycerol, and 0.1% bromophenol blue) to the mitochondrial RNA prepared in steps 3.1&#173;&#8211;3.3.</li>
	<li>Denature the RNA sample/Loading buffer mixture at 65 &#176;C for 10 min, and then chill on ice for 1 min.</li>
	<li>Prepare a denatured agarose gel (2% formaldehyde, 1.2% agarose, and 1x MOPS), and load the RNA sample/Loading buffer mixture to the gel (1&#8211;2 &#956;g mitochondrial RNA per well).</li>
	<li>Run the gel in 1x MOPS at 3&#8211;4 V/cm for ~4 h.</li>
	<li>Prepare 2 L of 20x SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0). Treat it with 0.1% DEPC overnight, and sterilize by autoclave.</li>
	<li>Rinse the gel twice in 20x SSC (15 min each time), and transfer gel RNA to a nylon membrane (Table of Materials) by capillary transfer with 20x SSC for 10&#8211;16 h.</li>
	<li>Fix the RNA to membrane by baking at 120 &#176;C for 30 min.</li>
	<li>Perform RNA hybridization and immunological detection with a commercial kit (Table of Materials) according to the manufacturer&#8217;s instruction.</li>
</ol>
11. Discrimination of Primary and Processed 5&#8217; Ends
<ol>
	<li>Discriminate the primary and processed 5&#8217; ends by comparing the cRT-PCR products obtained from the normalized 5&#8217; polyphosphatase-treated and non-treated RNAs. 
	NOTE: To primary transcripts, the abundance of PCR products from 5&#8217; polyphosphatase-treated RNA is much higher than that from non-treated counterpart (Figure 1, &#8216;Gene A,&#8217; and Figure 4A). However, to processed transcripts, a comparable level of PCR products would be amplified from the two sets of mitochondrial RNAs (Figure 1, &#8216;Gene B&#8217;).</li>
	<li>Design RT-qPCR primers based on the cRT-PCR mapping results.</li>
	<li>Verify the cRT-PCR discrimination results by RT-qPCR. 
	NOTE: The two cDNA samples derived from 5&#8217; polyphosphatase-treated and non-treated RNAs are normalized by the RT-qPCR products of 26S mature rRNA.</li>
</ol>

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M., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , (2012).</li><li>Canino, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+encodes+four+tRNase+Z+enzymes.">Arabidopsis encodes four tRNase Z enzymes.</a> Plant Physiology. 150 (3), 1494-1502 (2009).</li><li>Gobert, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+Arabidopsis+organellar+protein+has+RNase+P+activity.">A single Arabidopsis organellar protein has RNase P activity.</a> Nature Structural, Molecular Biology. 17, 740-744 (2010).</li><li>Gutmann, B., Gobert, A., Giege, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRORP+proteins+support+RNase+P+activity+in+both+organelles+and+the+nucleus+in+Arabidopsis.">PRORP proteins support RNase P activity in both organelles and the nucleus in Arabidopsis.</a> Genes &amp; Development. 26 (10), 1022-1027 (2012).</li><li>Stern, D. 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The authors have nothing to disclose.

In a previous study, total and mitochondrial RNAs from cell suspension culture of Arabidopsis were used to map mitochondrial transcript termini by cRT-PCR, and similar results were obtained12. However, only enriched mitochondrial RNA was used to map mitochondrial transcript termini in many other studies1,2,3,9. We found that the enrichment of mitochondrial RNA i...

In plant mitochondria, many mature and precursor RNAs are accumulated as multiple isoforms, and the steady-state transcripts can be divided into two groups based on the difference at their 5&#39; ends1,2,3,4. The primary transcripts have 5&#39; triphosphate ends, which are derived from transcription initiation. By contrast, the processed transcripts have 5&#39; monophosphate generated by post-transcriptional processing. Discrimination and mapping of the two types of transcripts are important to unravel the molecular mechanisms underlying transcription and transcript end maturation.
To distinguish between the primary and processed transcripts in plant mitochondrion, four major strategies have been developed. The first strategy is to pre-treat the mitochondrial RNAs with tobacco acid pyrophosphatase (TAP), which converts 5&#39; triphosphate to monophosphate and enables primary transcripts to be circularized by RNA ligase. The transcript abundances of TAP-treated and non-treated RNA samples are then compared by rapid amplification of cDNA ends (RACE) or circular RT-PCR (cRT-PCR)2,3,4. In the second strategy, processed transcripts are firstly depleted from mitochondrial RNAs using terminator 5&#39;-phosphate-dependant exonuclease (TEX), and the primary transcripts left are then mapped by primer extension analysis5,6. The third strategy is to pre-cap the primary transcripts using guanylyl transferase, and then the position of triphosphated 5&#39; termini is determined by primer extension together with ribonuclease or S1 nuclease protection analysis7,8,9. Different from those depending on the presence/absence of 5&#39; triphosphate, the fourth strategy combines in vitro transcription, site-directed mutagenesis, and primer extension analysis to characterize the putative promoters and determine the transcription initiation sites8,10,11. By using these strategies, many primary and processed transcripts have been determined in plant mitochondria.
However, several studies have reported that the 5&#39; triphosphate of primary transcripts were unstable, and they were easily converted to monophosphate for unknown reason2,4,12,13. This problem hinders a clear discrimination of the two types of transcripts by using techniques depending on the presence/absence of 5&#39; triphosphate, and previous efforts to systematically discriminate between the primary and processed transcripts in plant mitochondria failed2,12.
In this protocol, we combine cRT-PCR, RNA 5&#39; polyphosphatase treatment, RT-qPCR, and Northern blot to systematically distinguish the primary and processed transcripts stably accumulated in maize (Zea mays) mitochondrion (Figure 1). cRT-PCR allows simultaneous mapping of 5&#39; and 3&#39; extremities of a RNA molecule, and it is usually adapted to map transcript termini in plants2,12,14,15. RNA 5&#39; polyphosphatase could remove two phosphates from the triphosphated 5&#39; termini, which makes the primary transcripts available for self-ligation by RNA ligase. Previous studies showed that mature 26S rRNA in maize had processed 5&#39; terminus, and it was insensitive to RNA 5&#39; polyphosphatase1,16. To minimize the influence of unstable triphosphate at primary 5&#39; termini, the 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by mature 26S rRNA, and the primary and processed transcripts are then differentiated by comparing the cRT-PCR products obtained from the two RNA samples. The cRT-PCR mapping and discrimination results are verified by Northern blot and RT-qPCR, respectively. Finally, alternative primers are used to amplify those transcripts detected in Northern blot but not by cRT-PCR. By using this cRT-PCR-based strategy, most steady-state transcripts in maize mitochondrion have been differentiated and mapped1.

<table>
	<tbody>
		<tr>
			<td>Acetic acid</td>
			<td>Aladdin, China</td>
			<td>A112880</td>
			<td>To prepare 1x TAE buffer</td>
		</tr>
		<tr>
			<td>Applied Biosystems 2720 Thermal Cycler</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>4359659</td>
			<td>Thermal cycler for PCR amplification</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900134</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Biowest Agarose</td>
			<td>Biowest, Spain</td>
			<td>9012-36-6</td>
			<td>To resolve PCR products and RNAs</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-aldrich, USA</td>
			<td>A1933</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-aldrich, USA</td>
			<td>B8026</td>
			<td>For preparation of loading buffer for agarose gel electrophoresis and Northern blot</td>
		</tr>
		<tr>
			<td>DEPC</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900882</td>
			<td>Deactivation of RNase</td>
		</tr>
		<tr>
			<td>DIG Northern starter kit</td>
			<td>Roche, USA</td>
			<td>12039672910</td>
			<td>For DIG-RNA labeling and Northern blot. This kit contains the reagents for transcription-labeling of RNA with DIG and T7 RNA polymerase, hybridization and chemiluminescent detection.</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900106</td>
			<td>For preparation of extraction buffer and 1x TAE buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-aldrich, USA</td>
			<td>E3889</td>
			<td>For preparation of wash buffer</td>
		</tr>
		<tr>
			<td>Gel documentation system</td>
			<td>Bio-Rad, USA</td>
			<td>Gel Doc XR+</td>
			<td>To image the agarose gel</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-aldrich, USA</td>
			<td>G5516</td>
			<td>For preparation of loading buffer for agarose gel electrophoresis</td>
		</tr>
		<tr>
			<td>GoldView II (5000x)</td>
			<td>Solarbio,. China</td>
			<td>G8142</td>
			<td>DNA staining</td>
		</tr>
		<tr>
			<td>Hybond-N+, Nylon membrane</td>
			<td>Amersham Biosciences, USA</td>
			<td>RPN119</td>
			<td>For Northern blot</td>
		</tr>
		<tr>
			<td>Image Lab</td>
			<td>Bio-Rad, USA</td>
			<td>Image Lab 3.0</td>
			<td>Image gel, and compare the abundance of PCR products.</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900041</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>KOH</td>
			<td>Aladdin, China</td>
			<td>P112284</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900399</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Millex</td>
			<td>Millipore, USA</td>
			<td>SLHP033RB</td>
			<td>To sterile extraction and wash buffers by filtration</td>
		</tr>
		<tr>
			<td>Miracloth</td>
			<td>Calbiochem, USA</td>
			<td>475855-1R</td>
			<td>To filter the ground kernel tissues</td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900306</td>
			<td>For preparation of running buffer for Northern blot</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>2000C</td>
			<td>For RNA concentration and purity assay</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900797</td>
			<td>For preparation of wash buffer</td>
		</tr>
		<tr>
			<td>pEASY-Blunt simple cloning vector</td>
			<td>TransGen Biotech, China</td>
			<td>CB111</td>
			<td>Cloning of the gel-recovered band. It contains a T7 promoter several bps upstream of the insertion site.</td>
		</tr>
		<tr>
			<td>Phanta max super-fidelity DNA polymerase</td>
			<td>Vazyme, China</td>
			<td>P505</td>
			<td>DNA polymerase for PCR amplification</td>
		</tr>
		<tr>
			<td>Polyvinylpyrrolidone 40</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900008</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>Primer Premier 6.24</td>
			<td>PREMIER Biosoft, USA</td>
			<td>Primer Premier 6.24</td>
			<td>To design primers for reverse transcription and PCR amplification</td>
		</tr>
		<tr>
			<td>PrimeScript II reverse transcriptase</td>
			<td>Takara, Japan</td>
			<td>2690</td>
			<td>To synthesize the first strand cDNA</td>
		</tr>
		<tr>
			<td>PureLink RNA Mini kit</td>
			<td>Thermo Fisher Scientific, USA</td>
			<td>12183025</td>
			<td>For RNA purificaion</td>
		</tr>
		<tr>
			<td>RNA 5&#39; polyphosphatase</td>
			<td>Epicentre, USA</td>
			<td>RP8092H</td>
			<td>To convert 5&#39; triphosphate to monophosphate</td>
		</tr>
		<tr>
			<td>RNase inhibitor</td>
			<td>New England Biolabs, UK</td>
			<td>M0314</td>
			<td>A component of RNA self-ligation and 5&#39; polyphosphatase treatment reactions, and it is used to inhibite the activity of RNase.</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900212</td>
			<td>For preparation of running buffer for Northern blot</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-aldrich, USA</td>
			<td>V900058</td>
			<td>To prepare 20x SSC</td>
		</tr>
		<tr>
			<td>SsoFas evaGreen supermixes</td>
			<td>Bio-Rad, USA</td>
			<td>1725202</td>
			<td>For RT-qPCR</td>
		</tr>
		<tr>
			<td>T4 RNA Ligase 1</td>
			<td>New England Biolabs, UK</td>
			<td>M0437</td>
			<td>For RNA circularization</td>
		</tr>
		<tr>
			<td>Tetrasodium pyrophosphate</td>
			<td>Sigma-aldrich, USA</td>
			<td>221368</td>
			<td>For preparation of extraction buffer</td>
		</tr>
		<tr>
			<td>TIANgel midi purification kit</td>
			<td>Tiangen Biotech, China</td>
			<td>DP209</td>
			<td>To purify DNA fragments from agarose gel</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Aladdin, China</td>
			<td>T110601</td>
			<td>To prepare 1x TAE buffer</td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Invitrogen, USA</td>
			<td>15596026</td>
			<td>To extract mitochondiral RNA.</td>
		</tr>
		<tr>
			<td>Universal DNA purification kit</td>
			<td>Tiangen Biotech, China</td>
			<td>DP214</td>
			<td>To recover linearized plastmids from the restriction enzyme digestion reaction</td>
		</tr>
		<tr>
			<td>Xylene cyanol FF</td>
			<td>Sigma-aldrich, USA</td>
			<td>X4126</td>
			<td>For preparation of loading buffer for agarose gel electrophoresis</td>
		</tr>
	</tbody>
</table>

discrimintion and mapping of the primary and processed transcripts in maize mitochondrion using a circular rt-pcr-based strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

Estimation of mitochondrial RNA circularization efficiency
In a previous study, both total and mitochondrial RNAs were used for cRT-PCR mapping of mitochondrial transcript termini in Arabidopsis (Arabidopsis thaliana), and the two types of RNAs gave similar mapping results12. Initially, we also used total RNAs for cRT-PCR mapping of mitochondrial transcript termini in ma...

Watch this Scientific Journal Video about Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy at JoVE.com

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

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Research

JoVE Journal

Genetics

6.1K Views.  South China Agricultural University. We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5' polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5' triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

Video: Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the identification and verification of a drug resistance gene in the Apicomplexan parasite Toxoplasma gondii, the method of Quantitative Trait Locus (QTL) mapping using a Whole Genome Sequence (WGS) -based genetic map and the method of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 -based gene editing. The approach of QTL mapping allows one to test if there is a correlation between a genomic region(s) and a phenotype. Two datasets are required to run a QTL scan, a genetic map based on the progeny of a recombinant cross and a quantifiable phenotype assessed in each of the progeny of that cross. These datasets are then formatted to be compatible with R/qtl software that generates a QTL scan to identify significant loci correlated with the phenotype. Although this can greatly narrow the search window of possible candidates, QTLs span regions containing a number of genes from which the causal gene needs to be identified. Having WGS of the progeny was critical to identify the causal drug resistance mutation at the gene level. Once identified, the candidate mutation can be verified by genetic manipulation of drug sensitive parasites. The most facile and efficient method to genetically modify T. gondii is the CRISPR/Cas9 system. This system comprised of just 2 components both encoded on a single plasmid, a single guide RNA (gRNA) containing a 20 bp sequence complementary to the genomic target and the Cas9 endonuclease that generates a double-strand DNA break (DSB) at the target, repair of which allows for insertion or deletion of sequences around the break site. This article provides detailed protocols to use CRISPR/Cas9 based genome editing tools to verify the gene responsible for sinefungin resistance and to construct transgenic parasites.

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

Details are presented on how QTL mapping with a whole genome sequence based genetic map can be used to identify a drug resistance gene in Toxoplasma gondii and how this can be verified with the CRISPR/Cas9 system that efficiently edits a genomic target, in this case the drug resistance gene.

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration of transgenes. However, due to the low efficiency of homologous recombination (HR) and various indel mutations of non-homologous end joining (NHEJ)-based strategies in non-dividing cells, in vivo genome editing remains a great challenge. Here, we describe a homology-mediated end joining (HMEJ)-based CRISPR/Cas9 system for efficient in vivo precise targeted integration. In this system, the targeted genome and the donor vector containing homology arms (~800 bp) flanked by single guide RNA (sgRNA) target sequences are cleaved by CRISPR/Cas9. This HMEJ-based strategy achieves efficient transgene integration in mouse zygotes, as well as in hepatocytes in vivo. Moreover, a HMEJ-based strategy offers an efficient approach for correction of fumarylacetoacetate hydrolase (Fah) mutation in the hepatocytes and rescues Fah-deficiency induced liver failure mice. Taken together, focusing on targeted integration, this HMEJ-based strategy provides a promising tool for a variety of applications, including generation of genetically modified animal models and targeted gene therapies.

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system provides a promising tool for genetic engineering, and opens up the possibility of targeted integration of transgenes. We describe a homology-mediated end joining (HMEJ)-based strategy for efficient DNA targeted integration in vivo and targeted gene therapies using CRISPR/Cas9.

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

Adapting 3&#039; Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...