We thank Maya Wilson and Aneika Leney for the critical reading of the manuscript. This work was supported by a Sir Henry Dale Fellowship jointly funded by the Wellcome Trust and the Royal Society (200473/Z/16/Z).

1. Strains, Media, and Buffer Recipes
<ol>
	<li>Prepare yeast (S. cerevisiae) media as detailed here: YP (1% w/v yeast extract, 2% w/v bacteriological peptone), and&#160;glucose and galactose stocks at 20% w/v.</li>
	<li>Prepare sodium acetate (NaAc)-EDTA (AE) buffer as detailed here: 50 mM NaAc pH 5.3 and 10 mM EDTA.</li>
	<li>Prepare 10-23 DNAzyme 4x Incubation Buffer as detailed here: 24 mM Tris pH 8.0, 60 mM NaCl and 10-23 DNAzyme 4x Reaction Buffer: 200 mM Tris pH 8.0 and 600 mM NaCl.</li>
	<li>Prepare 8-17 DNAzyme 2x Reaction Buffer as detailed here: 200 mM KCl, 800 mM NaCl, 100 mM HEPES pH 7.0, 15 mM MgCl2, and 15 mM MnCl2.</li>
	<li>Prepare 10x MOPS Buffer as detailed here: 200 mM MOPS, 50 mM NaAc, 1 mM EDTA; pH 7.0 and 1.5x Sample Denaturing Buffer: 50% v/v formamide, 20% v/v formaldehyde, 1.5x MOPS buffer.</li>
	<li>Obtain S. cerevisiae strains, BY4741 (MATa his3&#916;1 leu2&#916;0 met15&#916;0 ura3&#916;0); GAL1::SNR13 (as BY4741 but GAL1::SNR13:KANmX); GAL1::SNR47 (as BY4741 but GAL1::SNR47:HIS3mX). Any other yeast strain can be used for this analysis.</li>
</ol>
2. DNAzyme Design
<ol>
	<li>Find RNA sequence of interest or putative methylation site using an appropriate database. For S. cerevisiae snoRNA targets, use yeast snoRNA database: http://people.biochem.umass.edu/fournierlab/snornadb/mastertable.php14</li>
	<li>To find the methylation site of interest, e.g., snR13-dependent site, select &#8220;snR13&#8221; and make a note of the position of the modified nucleotide (e.g., snR13-guided A2281 in 25S rRNA).</li>
	<li>Find sequences upstream and downstream of the modified nucleotide using appropriate database. For S. cerevisiae, use the Saccharomyces Genome Database: https://www.yeastgenome.org/</li>
	<li>Search for the target gene name e.g., RDN25 (coding 25S rRNA).</li>
	<li>From the &#8220;sequence&#8221; tab, select 10-15 nucleotides upstream (5&#8217; arm) and downstream (3 &#8217;arm) of the methylation site when using a 10-23 DNAzyme assay and 20 nucleotides upstream (5&#8217; arm) and downstream (3&#8217;arm) of the methylation site for a 8-17 DNAzyme.</li>
	<li>Create complementary sequences of 5&#8217; and 3&#8217; arms.</li>
	<li>Flank 10-23 or 8-17 DNAzyme catalytic sequence with complementary sequences of 5&#8217; and 3&#8217; arms.</li>
	<li>Order DNAzyme as a normal DNA oligonucleotide from the supplier.</li>
</ol>
3. S. cerevisiae Growth Conditions
NOTE: S. cerevisiae BY4741 strain derivatives were used, in which the expression of either SNR13 or SNR47 snoRNA is driven from the inducible GAL1 promoter. In order to induce or inhibit their synthesis, grow cells on medium containing galactose (GAL1-dependent transcription on) or glucose (GAL1-dependent transcription off). As a control, use the wild type strain (BY4741) grown either on galactose or glucose.
<ol>
	<li>Grow yeast strains in an appropriate medium and conditions. To analyze GAL1::SNR13 and GAL1::SNR47 strains as well as the isogenic wild type strain, grow cells in 50 mL of YP media with either 2% glucose (YPD) or galactose (YPGal) at 30 &#176;C to the middle exponential phase.</li>
	<li>Centrifuge cells at 1,000 x g, for 3 min at 4 &#176;C.</li>
	<li>Discard the supernatant and keep the pellets.</li>
	<li>Freeze cell pellets in liquid nitrogen and store them at -80 &#176;C. 
	CAUTION: Liquid nitrogen may cause severe cryogenic burns. Always wear protective clothing and exercise safety precautions. 
	NOTE: Cell pellets can be stored at -80 &#176;C up to 1 month. The protocol can be paused here if needed.</li>
</ol>
4. RNA Isolation15
NOTE: Use the most appropriate method to isolate RNA. For yeast S. cerevisiae, hot-phenol RNA extraction can be used.
<ol>
	<li>Add 1 mL of ice-cold water, resuspend the pellets and transfer resuspended cells to 1.5 mL microtubes.</li>
	<li>Centrifuge at 20,000 x g for 10 s at 4 &#176;C and remove the supernatant.</li>
	<li>Add 400 &#181;L of AE buffer and resuspend the cells. 
	NOTE: Steps 4.4-4.15 are performed at room temperature unless stated otherwise.</li>
	<li>Add 40 &#181;L of 10% SDS and 400 &#181;L of acid phenol (pH 4.5). 
	CAUTION: Phenol is toxic and should be handled under a fume hood. Always wear a lab coat, protective gloves, and glasses when working with phenol. Dispose of the waste according to the institutional regulations.</li>
	<li>Mix well by vortexing for 20 s.</li>
	<li>Incubate at 65 &#176;C for 10 min. Every 2 min, gently open and close the tube to release the pressure and flip the tube 2-3 times to mix the phases.</li>
	<li>Transfer the tubes to -80 &#176;C and incubate for 10 min.</li>
	<li>Defrost the tubes on the bench and centrifuge at 20,000 x g, for 5 min at room temperature.</li>
	<li>Transfer the upper phase to a new tube containing 400 &#181;L acid phenol:chloroform:isoamyl alcohol (25:24:1). Do not disrupt the interphase. 
	CAUTION: Chloroform is toxic and should be handled under a fume hood. Always wear a lab coat, protective gloves, and glasses when working with chloroform. Dispose of the waste according to institutional regulations.</li>
	<li>Mix well by vortexing for 30 s and centrifuge at 20,000 x g for 10 min at room temperature.</li>
	<li>Transfer the upper phase (~400 &#181;L) to a new tube containing 400 &#181;L chloroform.</li>
	<li>Mix well by vortexing for 30 s and centrifuge at 20,000 x g for 5 min at room temperature.</li>
	<li>Transfer the upper phase (~300-350 &#181;L) to a new tube containing 1 mL of EtOH and 40 &#181;L of 7.5 M ammonium acetate (NH4AC). Mix by flipping the tube a few times.</li>
	<li>Incubate at -80 &#176;C for 2 h or overnight at -20 &#176;C. 
	NOTE: The procedure can be paused here.</li>
	<li>Centrifuge at 20,000 x g, for 10 min at 4 &#176;C. A small, white RNA pellet will become visible on the bottom of the tube.</li>
	<li>Remove EtOH by pipetting to avoid disturbing the pellet.</li>
	<li>Add 1 mL of 70% EtOH and centrifuge at 20,000 x g for 5 min at room temperature.</li>
	<li>Remove 70% EtOH by pipetting.</li>
	<li>Centrifuge at 20,000 x g for 15 s and remove the remaining EtOH with 2-20 &#181;L pipette.</li>
	<li>Leave the tube open on the bench for 5 min to dry the RNA pellet. 
	NOTE: RNA pellet changes its color from white to transparent when dry.</li>
	<li>Resuspend the RNA pellet in 30 &#181;L of RNase/DNase-free H2O, transfer the tube immediately on the ice and measure RNA concentration on microspectrophotometer.</li>
	<li>Freeze the samples at -20 &#176;C. 
	NOTE: RNA can be stored at -20 &#176;C up to 1 month and at -80 &#176;C up to 1 year. The procedure can be paused here or proceeded directly to the next step.</li>
</ol>
5. DNAzyme Digestion
<ol>
	<li>10-23 DNAzyme digestion
	<ol>
		<li>In 1.5 mL tubes prepare an incubation mix by combining 5 &#181;g of RNA, 200 pmol of 10-23 DNAzyme (2 &#181;L of 100 &#956;M stock solution) and 2.5 &#181;L of 4x 10-23 Incubation Buffer in a total volume of 10 &#181;L. Keep the tubes on ice.</li>
		<li>Transfer the tubes to a dry heat block set at 95 &#176;C and incubate for 3 min.</li>
		<li>Transfer the tubes immediately on ice and incubate for 5 min.</li>
		<li>Spin down briefly and put the tubes back on the ice.</li>
		<li>Add 20 U of RNase inhibitor (e.g., 0.5 &#181;L RiboLock RNase inhibitor).</li>
		<li>Place the tubes in a dry heat block set for 25 &#176;C and incubate for 10 min.</li>
		<li>In the meantime, prepare a reaction mixture in a 1.5 &#181;L tube by combining 5 &#181;L of 4x 10-23 Reaction Buffer with 4 &#181;L of 300 mM MgCl2 and 1 &#181;L H2O. Place the tube in a dry block set to 37 &#176;C.</li>
		<li>Transfer the incubation mix to a dry heat block set for 37 &#176;C and add 10 &#181;L of pre-warmed reaction mix.</li>
		<li>Incubate the reaction at 37 &#176;C for 1 h.</li>
		<li>Transfer the tubes on ice and proceed to step 5.3.1.</li>
	</ol>
	</li>
	<li>8-17 DNAzyme digestion
	<ol>
		<li>Prepare a 1.5 mL microtube with 5 &#181;g of RNA in a total volume of 6 &#181;L. Keep the tube on ice.</li>
		<li>Prepare a 1.5 mL microtube with 400 pmol of 8-17 DNAzyme (4 &#181;L out of 100 &#956;M stock). Keep the tube on ice.</li>
		<li>Transfer the tubes to a dry heat block set for 95 &#176;C and incubate for 2 min.</li>
		<li>Move the RNA sample on ice.</li>
		<li>Spin down the tube with DNAzyme for 5 s and incubate at 25 &#176;C for 10 min.</li>
		<li>At the same time, prepare a 1.5 mL tube with 10 &#181;L of 2x 8-17 Reaction Buffer and incubate at 25 &#176;C.</li>
		<li>Prepare a reaction mixture by adding 10 &#181;L of pre-warmed 2x Reaction Buffer to the tube with DNAzyme.</li>
		<li>Transfer 14 &#181;L of the reaction mix to the tube with RNA and add 20 U of RNase inhibitor.</li>
		<li>Incubate the reaction at 25 &#176;C for 2 h.</li>
		<li>Transfer the tube on ice and proceed to RNA purification (step 5.3.1).</li>
	</ol>
	</li>
	<li>RNA purification
	<ol>
		<li>Add 350 &#181;L of water and 400 &#181;L of chloroform to the reaction tube, mix well by vortexing for 30 s and centrifuge at 20,000 x g for 5 min at room temperature.</li>
		<li>Transfer the upper phase (~300-350 &#181;L) to a new tube containing 1 mL of EtOH, 40 &#181;L of 7.5 M NH4AC and 1 &#181;L of glycogen (10 &#181;g/&#181;L). Mix by flipping the tube a few times.</li>
		<li>Incubate at -80 &#176;C for 2 h or overnight at -20 &#176;C. 
		NOTE: The procedure can be paused here.</li>
		<li>Repeat steps from 4.15 to 4.21.</li>
		<li>Resuspend the RNA pellet in 10 &#181;L of RNase/DNase-free H2O and transfer the tubes immediately on ice.</li>
		<li>Freeze the samples at -20 &#176;C. 
		NOTE: RNA can be stored at -20 &#176;C up to a month and at -80 &#176;C up to 1 year. The procedure can be paused here or proceed to RNA electrophoresis.</li>
	</ol>
	</li>
</ol>
6. RNA Electrophoresis
<ol>
	<li>Spray the electrophoresis equipment (tank, tray, comb) with 1% SDS, leave for 15 min and rinse with plenty of ddH2O.</li>
	<li>Dissolve 1.5 g of agarose in 127.5 mL of ddH2O by heating it in the microwave.</li>
	<li>Add 15 mL of 10x MOPS and 7.5 mL of 37% formaldehyde to the agarose solution (total volume is 150 mL). 
	CAUTION: Formaldehyde is toxic and should be handled under a fume hood. Always wear a lab coat, protective gloves, and glasses when working with formaldehyde. Dispose of the waste according to institutional regulations.</li>
	<li>Add an appropriate amount of a gel stain of choice to the agarose solution (e.g., 15 &#181;L of SYBR Safe DNA gel stain). Mix well and pour the agarose to the tray.</li>
	<li>Insert a comb in the gel immediately.</li>
	<li>Leave it for 45 min under the fume hood. Cover the tray with aluminum foil when using a light-sensitive gel stain.</li>
	<li>Prepare 600 mL of 1x MOPS buffer.</li>
	<li>RNA sample preparation
	<ol>
		<li>In a 1.5 mL tube, combine 10 &#181;L of the digested and purified RNA sample, 5 &#181;L of Sample Denaturing Buffer and 0.5 &#181;L of 6x Loading Dye. 
		CAUTION: Formamide is toxic and should be handled under a fume hood. Always wear a lab coat, protective gloves, and glasses when working with formamide. Dispose of the waste according to institutional regulations.</li>
		<li>Incubate RNA samples at 70 &#176;C for 5 min. Transfer the samples on ice. Incubate for 5 min.</li>
		<li>Spin down briefly before loading on the gel.</li>
	</ol>
	</li>
	<li>Put the gel in the electrophoresis tank and fill with 1x MOPS buffer. Load the entire volume of each sample (15 &#181;L) on the gel. Run at 80 V until bromophenol blue reaches 2/3 of the gel length.</li>
	<li>Image the gel using an imager appropriate to detect the chosen gel stain (e.g., UV transilluminator).</li>
</ol>

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The authors have nothing to disclose.

DNAzyme-dependent digestion can be used as a simple and quick method to analyze site-specific RNA 2&#8217;-O-methylation12,13. DNAzymes cleave RNA if the nucleotide upstream of the cleavage site is not methylated. In contrast to other approaches, including RNase H-directed digestion, alkaline degradation or reverse transcription in low nucleotides concentration followed by quantitative PCR or sequencing8,10,11,16, DNAzyme approach requires a simple DNA oligonucleotide and basic reagents that are present in any molecular biology laboratory. Moreover, DNAzymes may be used in a similar way to analyze RNA pseudouridylation mediated by box H/ACA snoRNA12, which makes them versatile tools in studying snoRNA targets.
DNAzyme-dependent approaches are limited only by cleavage site consensus sequences17. 10-23 DNAzymes can be used to analyze 2&#8217;-O-methylation only at position R of the RY dinucleotide, while 8-17 DNAzymes recognize the modification of the nucleotide located upstream of guanine. As a result, modifications like 2&#8217;-O-methylation of the first nucleotide in the dinucleotides guanine-adenine (GA), adenine-adenine (AA), pyrimidine-adenine (YA) and pyrimidine-pyrimidine (YY) cannot be analyzed. Moreover, the low efficiency of DNAzyme-dependent cleavage12 should be considered. Although some DNAzymes cleave RNA almost completely (Figure 3B), many DNAzymes only partially digest their targets (Figure 3B). The efficiency may depend on the sequence surrounding the cleavage site. For example, RNA regions with stretches of the same nucleotide may affect the correct positioning of the DNAzyme active sequence. Furthermore, RNA regions forming strong secondary structure may re-hybridize and suppress DNAzyme binding to the target sequence. To overcome these issues, cycles of heating and cooling of the 10-23 DNAzyme and its RNA substrate can be applied18.
We used the DNAzyme approach to investigate 2&#8217;-O-methylation of rRNA. One can also &#160;use this technique to analyze other RNA modifications, such as N6-methyladenosine19. Ribosomal RNA, due to its abundance, can be analyzed by electrophoresis and the cleavage products can be visualized under the UV light. However, this is not applicable for less abundant RNAs like RNA Polymerase II-generated coding RNAs (mRNA) and non-coding RNAs (ncRNA). These RNAs cannot usually be detected directly by RNA staining in agarose or polyacrylamide gels. In such cases, DNAzyme-dependent cleavage can be visualized by Northern blotting, indirectly detected by PCR/quantitative PCR or analyzed by quantitative PCR with polymerases (e.g., KlenTaq DNA Polymerase) capable of discriminating 2&#8242;-O-methylated RNA from unmethylated RNA20,21.

RNA modifications play important roles in the regulation of gene expression. RNA 2&#8217;-O-methylation and pseudouridylation, which are guided by box C/D and box H/ACA small nucleolar RNAs (snoRNAs) respectively, protect RNA from degradation and stabilize their higher-order structures1,2,3. SnoRNA targets have been identified mainly in ribosomal RNAs (rRNA) and small nuclear RNAs (snRNAs). However, in higher eukaryotes, there are potentially hundreds of snoRNA with no assigned functions and some of them may recognize multiple RNAs1,4,5,6,7. Therefore, methods which allow for the identification and analysis of snoRNA-guided modifications are important tools in uncovering mechanisms governing cellular processes.
A box C/D snoRNA-guided putative 2&#8217;-O-methylation site can be identified bioinformatically and confirmed experimentally by many techniques, including RNase H-directed cleavage, or site-specific and genome-wide methods, which employ reverse transcription in low nucleotides (dNTPs) concentration approach8,9,10,11. These techniques are very sensitive but also laborious and expensive, therefore, may not be suitable for the initial or quick testing. One of the simplest and low-cost methods to identify 2&#8217;-O-methylation sites is DNAzyme-dependent RNA cleavage12. DNAzymes are short, single-stranded and catalytically active DNA molecules capable of endonucleolytic cleavage of RNA at specific positions. They consist of a conserved and catalytically active core sequence and 5&#8217; and 3&#8217; binding arms composed of variable sequences designed to hybridize by Watson-Crick base-pairing to the RNA target (Figure 1). Thus, the 5&#8217; and 3&#8217; arms deliver the catalytic sequence to the specific RNA site. DNAzyme-dependent cleavage is inhibited by 2&#8217;-O-methylation of the nucleotide positioned directly upstream of the cleavage site12,13. This makes DNAzymes very practical tools for the analysis of putative or known RNA 2&#8217;-O-methylation sites.
Two types of DNAzymes are used for RNA modifications analyses12. The active sequence of 10-23 DNAzyme (Figure 1A) consists of 15 nucleotides (5&#8217;RGGCTAGCTACAACGA3&#8217;) which form a loop around the targeted RNA purine-pyrimidine (RY) dinucleotide and catalyze the cleavage between these two nucleotides. The RNA purine (R) is not base-paired with the DNAzyme and the 2&#8217;-O-methylation presents on the DNAzyme inhibits the cleavage. The binding arms of 10-23 DNAzymes are usually 10-15 nucleotides long. The second DNAzyme class, 8-17 DNAzymes (Figure 1B) contain 14-nucleotide catalytic sequence (5&#8217;TCCGAGCCGGACGA3&#8217;). Nucleotides C2, C3 and G4 pair with C9 G10 and G11 forming a short stem-loop structure. 8-17 DNAzymes cleave RNA upstream of any guanine that is imperfectly paired with the first thymine from the DNAzyme active sequence. The RNA nucleotide upstream of the guanine is not base-paired with DNAzyme and its 2&#8217;-O-methylation impairs the cleavage. 8-17 DNAzymes require longer homology arms of around 20 nucleotides to direct DNAzyme to its specific sequence.
Here, we provide a step-by-step protocol for the analysis of 2&#8217;-O-methylation of rRNA in Saccharomyces cerevisiae using 10-23 and 8-17 DNAzyme-dependent approaches12,13 (Figure 1C). This protocol can be easily adapted for other organisms and RNA species and employed for the fast, preliminary or major analyses of site-specific RNA 2&#8217;-O-methylation.

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acid phenol</td>
			<td>SIGMA</td>
			<td>P4682</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>VWR</td>
			<td>A2114</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>SIGMA</td>
			<td>A1542</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorophorm</td>
			<td>Fisher scientific</td>
			<td>10293850</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase/RNase free water</td>
			<td>Fischer Scientific</td>
			<td>10526945</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAzyme</td>
			<td>Integrated DNA Technology</td>
			<td>Custom oligo DNA</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol Absolute</td>
			<td>Fisher scientific</td>
			<td>10437341</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>sigma</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>Galactose</td>
			<td>SIGMA</td>
			<td>G0750</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Loading Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0611</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>SIGMA</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Fisher Scientific</td>
			<td>R0561</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>SIGMA</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoamyl</td>
			<td>SIGMA</td>
			<td>W205702</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>SIGMA</td>
			<td>P9333</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>SIGMA</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>SIGMA</td>
			<td>244589</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>SIGMA</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>SIGMA</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxoid Peptone Bacteriological</td>
			<td>Thermo Fisher Scientific</td>
			<td>LP0037</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxoid Yeast Extract Powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>LP0021</td>
			<td></td>
		</tr>
		<tr>
			<td>RiboLock RNase Inhibitor (40 U/&#181;L)</td>
			<td>Thermo Fisher Scientific</td>
			<td>EO0382</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>SIGMA</td>
			<td>74255</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate trihydrate</td>
			<td>SIGMA</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA Gel Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>SIGMA</td>
			<td>TRIS-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microtubes</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>152VR5C01M -80&#176;C freezer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Erlenmeyer flasks</td>
			<td>Cole-Parmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>Sarstedt</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Combicup VX200 vortex</td>
			<td>Appleton Woods</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DS-11 microspectrophotometer</td>
			<td>Denovix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Electrophoresis chamber (20 cm tray)</td>
			<td>SIGMA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FiveEasy F20 pH meter</td>
			<td>Appleton Woods</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gel documentation system</td>
			<td>Syngene</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Heraeus Fresco 21 micro centrifuge</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Megafuge 8R centrifuge with rotator suitable for 50 mL conical tubes</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Fuge Plus mini centrifuge</td>
			<td>Starlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mixer HC thermal block</td>
			<td>Starlab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>OLS26 Shaking Water Bath</td>
			<td>Grant</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac power supplier</td>
			<td>BioRad</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

dnazyme-dependent analysis of rrna 2&#8217;-o-methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

The utility of the DNAzyme-dependent cleavage in the analysis of rRNA modifications has been shown recently in the context of snoRNAs maturation13. The DNAzyme-dependent assay was used to show that lack of 5&#8217;-end pre-snoRNA processing affects 2&#8217;-O-methylation levels of 25S and 18S rRNA in S. cerevisiae13.
Here, we used an inducible snoRNA transcription system to demonstrate the effectiveness and simplicity of the technique. Box C/D snR13 guides methylation at two positions in 25S rRNA, including adenine 2281 (Figure 2A). This nucleotide is followed by uracil, which constitutes the consensus dinucleotide (RY) cleavable by a 10-23 DNAzyme. Box C/D snR47 also guides methylation of two nucleotides in 25S rRNA (Figure 2B). The adenine in position 2220 is followed by a guanine residue and this dinucleotide can be cleaved by an 8-17 DNAzyme. In order to induce or inhibit synthesis of either snR13 or snR47 snoRNA, we inserted the inducible GAL1 promoter upstream of either SNR13 or SNR47 genes and cultivated cells in medium containing galactose (GAL1-dependent transcription on) or glucose (GAL1-dependent transcription off). Next, RNA isolated from GAL1::SNR13 cells were incubated with 10-23 DNAzyme designed to cleave 25S rRNA at snR13-dependent site, in between nucleotides 2281 and 2282 (Figure 2C). RNA from GAL1::SNR47 strain was treated with 8-17 DNAzyme targeting snR47-dependent site in between nucleotides 2220 and 2221 (Figure 2D). As a control, RNA from the wild-type BY4741 strain growing on either galactose or glucose was incubated with both DNAzymes. Electrophoresis of the DNAzyme-treated RNA revealed that 25S rRNA extracted from GAL1::SNR13 and GAL1::SNR47 strains growing on galactose (GAL) remained intact (Figure 3A,B; lanes 3). In contrast, RNA isolated from GAL1::SNR13 and GAL1::SNR47 cells growing on glucose (GLC) was digested by respective DNAzymes (Figure 3A,B; lanes 4). In both cases, the 25S rRNA band decreased and 5&#8217; and 3&#8217; cut-off cleavage products (A and B) were observed. This indicates that in GAL1::SNR13 and GAL1::SNR47 strains, 25S rRNA was 2&#8217;-O-methylated at snR13- or snR47-guided sites when galactose was used as a carbon source and these snoRNA were expressed. The lack of 25S rRNA methylation when snR13 or snR47 expression was shut off on glucose allowed for DNAzyme-dependent cleavage. No RNA digestion was observed for wild-type samples (Figure 3A,B; lanes 1 and 2), as the expression of snR13 and snR47 is galactose/glucose-independent in this strain. Therefore, rRNA was normally methylated and so resistant to DNAzymes activity.
Overall, our experiment shows that the cleavage activity of 10-23 (Figure 3A) and 8-17 (Figure 3B) DNAzymes correlated with the absence of box C/D snR13 or snR47, clearly indicating that these snoRNA are responsible for 25S rRNA 2&#8217;-O-methylation at particular sites.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59700/59700fig1.jpg" /> 
Figure 1: DNAzymes and their RNA substrates. (A) 10-23 DNAzymes cleave a purine-pyrimidine (RY) RNA dinucleotide. R in the RNA is not paired with DNAzyme, while Y is complementary to the R base in the DNAzyme. Methylation of the purine (R) in RNA suppresses DNAzyme-dependent cleavage. (B) 8-17 DNAzymes cleave RNA upstream of guanine that is imperfectly paired with the first thymine in the DNAzyme catalytic sequence. The nucleotide preceding guanine is not paired and its methylation protects from DNAzyme-dependent cleavage. RNA is shown in grey (apart from the methylation site), DNAzyme is shown in purple. N = any nucleotide, R = purine: adenine or guanine, Y = pyrimidine: cytosine or uracil; CH3- denotes RNA methylation. Base pairing within the DNAzyme active sequences is marked by dotted lines. A blue lightning bolt marks the cleavage site. (C) A flowchart showing the steps of a DNAzyme-dependent analysis. <a href="https://www.jove.com/files/ftp_upload/59700/59700fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59700/59700fig2.jpg" /> 
Figure 2: DNAzymes targeting snR13- and snR47-dependent methylation sites in 25S rRNA.&#160;(A, B) Browser screenshots showing snR13-dependent (A) and snR47-dependent (B) methylation sites in 25S rRNA (C) 25S rRNA sequence surrounding snR13-dependent methylation site (A2281) and 10-23 DNAzyme (shown in purple) designed to cleave RNA between A2281 and U2282. A2281 is not paired with the DNAzyme while U2282 forms a pair with the first nucleotide from the DNAzyme active sequence (marked by a blue line). A blue lightning bolt marks the cleavage. (D) 25S rRNA sequence surrounding snR47-dependent methylation site (A2220) and 8-17 DNAzyme (shown in purple) designed to cleave RNA between A2220 and G2221. A2220 is not hybridized with the DNAzyme while G2221 is imperfectly paired with thymine (denoted with a dashed line). A blue lightning bolt marks the cleavage site. <a href="https://www.jove.com/files/ftp_upload/59700/59700fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59700/59700fig3.jpg" /> 
Figure 3: Analysis of site-specific 2&#8217;-O-methylation of 25S rRNA using 10-23 and 8-17 DNAzyme-dependent assay. (A) Analysis of snR13-dependent 25S rRNA methylation using 10-23 DNAzyme. (B) Analysis of snR47-dependent 25S rRNA methylation using 8-17 DNAzyme. RNA was visualized staining in a denaturing agarose gel. Cleavage products A and B are marked by red arrows. WT = wild-type strain; GAL = galactose, GLC = glucose. <a href="https://www.jove.com/files/ftp_upload/59700/59700fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about DNAzyme-dependent Analysis of rRNA 2Ã¢â‚¬â„¢-O-Methylation at JoVE.com

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

dnazyme-dependent-analysis-of-rrna-2-o-methylation

Research

JoVE Journal

Developmental Biology

8.2K Views.  University of Birmingham. Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2’-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

Video: DNAzyme-dependent Analysis of rRNA 2’-O-Methylation

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often compromise the lives of those who suffer from these complex diseases. Detecting mitochondrial metabolic changes is vital to the understanding of mitochondrial disorders and mitochondrial responses to pharmacological agents. Although mitochondrial metabolism is at the core of metabolic regulation, the detection of subtle changes in mitochondrial metabolism may be hindered by the overrepresentation of other cytosolic metabolites obtained using whole organism or whole tissue extractions. 
Here we describe an isolation method that detected pronounced mitochondrial metabolic changes in Drosophila that were distinct between whole-fly and mitochondrial enriched preparations. To illustrate the sensitivity of this method, we used a set of Drosophila harboring genetically diverse mitochondrial DNAs (mtDNA) and exposed them to the drug rapamycin. Using this method we showed that rapamycin modifies mitochondrial metabolism in a mitochondrial-genotype-dependent manner. However, these changes are much more distinct in metabolomics studies when metabolites were extracted from mitochondrial enriched fractions. In contrast, whole tissue extracts only detected metabolic changes mediated by the drug rapamycin independently of mtDNAs.

Preparation of Mitochondrial Enriched Fractions for Metabolic Analysis in Drosophila

Mitochondria play central roles in the regulation of metabolism and homeostasis. Subtle changes in mitochondrial metabolism that affect organismal physiology could be difficult to detect in whole organism metabolomics studies. Here we describe an isolation method that enhances the detection of subtle metabolic shifts in Drosophila melanogaster.

Since mitochondria play roles in amino acid metabolism, carbohydrate metabolism and fatty acid oxidation, defects in mitochondrial function often ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of vasculogenesis has become a central readout of endothelial progenitor cell functionality, and therefore, several attempts have been made to improve both in vitro and in vivo vasculogenesis models. However, standard methods are limited in scope, with static measurements failing to capture many aspects of this highly dynamic process. Therefore, the goal of developing this novel protocol was to assess the kinetics of in vitro vasculogenesis in order to quantitate rates of network formation and stabilization, as well as provide insight into potential mechanisms underlying vascular dysfunction. Application of this protocol is demonstrated using fetal endothelial colony forming cells (ECFCs) exposed to maternal diabetes mellitus. Fetal ECFCs were derived from umbilical cord blood following birth, cultured, and plated in slides containing basement membrane matrix, where they underwent vasculogenesis. Images of the entire slide wells were acquired using time-lapse phase contrast microscopy over 15 hours. Images were analyzed for derivation of quantitative data using an analysis software called Kinetic Analysis of Vasculogenesis (KAV). KAV uses image segmentation followed by skeletonization to analyze network components from stacks of multi-time point phase contrast images to derive ten parameters (9 measured, 1 calculated) of network structure including: closed networks, network areas, nodes, branches, total branch length, average branch length, triple-branched nodes, quad-branched nodes, network structures, and the branch to node ratio. Application of this protocol identified altered rates of vasculogenesis in ECFCs obtained from pregnancies complicated by diabetes mellitus. However, this technique has broad implications beyond the scope reported here. Implementation of this approach will enhance mechanistic assessment and improve functional readouts of vasculogenesis and other biologically important branching processes in numerous cell types or disease states.

Kinetic Analysis of Vasculogenesis Quantifies Dynamics of Vasculogenesis and Angiogenesis In Vitro

Here, we present a protocol for time-lapse imaging and analysis of vasculogenesis in vitro using phase contrast microscopy coupled with the open source software, Kinetic Analysis of Vasculogenesis. This protocol can be applied to quantitatively assess the vasculogenic potential of numerous cell types or experimental conditions that model vascular disease.

Vasculogenesis is a complex process by which endothelial stem and progenitor cells undergo de novo vessel formation. Quantitative assessment of ...

Gait Analysis of Age-dependent Motor Impairments in Mice with Neurodegeneration

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. Specifically, gait analysis allows recapturing disease relevant phenotypes that are observed in human patients, especially in neurodegenerative diseases that affect motor abilities such as Parkinson&#39;s disease (PD), Alzheimer&#39;s disease (AD), amyotrophic lateral sclerosis (ALS), and others. In early studies along this line, the measurement of gait parameters was laborious and depended on factors that were hard to control (e.g., running speed, continuous running). The development of ventral plane imaging (VPI) systems made it feasible to perform gait analysis at a large scale, making this method a useful tool for the assessment of motor behavior in rodents. Here, we present an in-depth protocol of how to use kinematic gait analysis to examine the age-dependent progression of motor deficits in mouse models of neurodegeneration; mouse lines with decreased levels of endophilin, in which neurodegenerative damage progressively increases with age, are used as an example.

In this study, we demonstrate the use of kinematic gait analysis based on ventral plane imaging to monitor the subtle changes in motor coordination as well as the progression of neurodegeneration with advancing age in mouse models (e.g., endophilin mutant mouse lines).

Motor behavior tests are commonly used to determine the functional relevance of a rodent model and to test newly developed treatments in these animals. ...

Analysis of Epididymal Protein Synthesis and Secretion

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation and storage of spermatozoa. Such complexity arises due to the combined secretory and absorptive activity of the lining epithelial cells. Here, we describe the techniques for the analysis of epididymal protein synthesis and secretion by focusing on the model protein family of dynamin (DNM) mechanoenzymes; large GTPases that have the potential to regulate bi-directional membrane trafficking events. For the study of protein expression in epididymal tissue, we describe robust methodology for immunofluorescence labeling of target proteins in paraffin-embedded sections and the subsequent detection of the spatial distribution of these proteins via immunofluorescence microscopy. We also describe optimized methodology for the isolation and characterization of exosome like vesicles, known as epididymosomes, which are secreted into the epididymal lumen to participate in intercellular communication with maturing sperm cells. As a complementary approach, we also describe the immunofluorescence detection of target proteins in an SV40-immortalized mouse caput epididymal epithelial (mECap18) cell line. Moreover, we discuss the utility of the mECap18 cell line as a suitable in vitro model with which to explore the regulation of epididymal secretory activity. For this purpose, we describe the culturing requirements for the maintenance of the mECap18 cell line and the use of selective pharmacological inhibition regimens that are capable of influencing their secretory protein profile. The latter are readily assessed via harvesting of conditioned culture medium, concentration of secreted proteins via trichloroacetic acid/acetone precipitation and their subsequent analysis via SDS-PAGE and immunoblotting. We contend that these combined methods are suitable for the analysis of alternative epididymal protein targets as a prelude to determining their functional role in sperm maturation and/or storage.

Here, we report the immunofluorescence localization of dynamin to illustrate the protocols for the detection of proteins in paraffin-embedded mouse epididymal sections and those of an immortalized epididymal cell line (mECap18). We also describe the protocols for the isolation of secretory proteins from both epididymal fluid and conditioned cell media.

The mammalian epididymis generates one of the most complex intraluminal fluids of any endocrine gland in order to support the post-testicular maturation ...

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits are required to control this behavior. Failure to produce the rhythmic pattern of contractions can be indicative of neurological abnormalities. We previously found that defects in protein O-mannosylation, a posttranslational protein modification, affect the axon morphology of sensory neurons and result in abnormal coordinated muscle contractions in embryos. Here, we present a relatively simple method for recording and analyzing the pattern of peristaltic muscle contractions by live imaging of late stage embryos up to the point of hatching, which we used to characterize the muscle contraction phenotype of protein O-mannosyltransferase mutants. Data obtained from these recordings can be used to analyze muscle contraction waves, including frequency, direction of propagation and relative amplitude of muscle contractions at different body segments. We have also examined body posture and taken advantage of a fluorescent marker expressed specifically in muscles to accurately determine the position of the embryo midline. A similar approach can also be utilized to study various other behaviors during development, such as embryo rolling and hatching.

Live Imaging and Analysis of Muscle Contractions in Drosophila Embryo

Here, we present a method to record embryonic muscle contractions in Drosophila embryos in a non-invasive and detail-oriented manner.

Coordinated muscle contractions are a form of rhythmic behavior seen early during development in Drosophila embryos. Neuronal sensory feedback circuits ...

Analysis of Hematopoietic Stem Progenitor Cell Metabolism

Hematopoietic stem progenitor cells (HSPCs) have distinct metabolic plasticity, which allows them to transition from their quiescent state to a differentiation state to sustain demands of the blood formation. However, it has been difficult to analyze the metabolic status (mitochondrial respiration and glycolysis) of HSPCs due to their limited numbers and lack of optimized protocols for non-adherent, fragile HSPCs. Here, we provide a set of clear, step-by-step instructions to measure metabolic respiration (oxygen consumption rate; OCR) and glycolysis (extracellular acidification rate; ECAR) of murine bone marrow-LineagenegSca1+c-Kit+ (LSK) HSPCs. This protocol provides a higher amount of LSK HSPCs from murine bone marrow, improves the viability of HSPCs during incubation, facilitates extracellular flux analyses of non-adherent HSPCs, and provides optimized injection protocols (concentration and time) for drugs targeting oxidative phosphorylation and glycolytic pathways. This method enables the prediction of the metabolic status and the health of HSPCs during blood development and diseases.

Hematopoietic stem progenitor cells (HSPCs) transition from a quiescent state to a differentiation state due to their metabolic plasticity during blood formation. Here, we present an optimized method for measuring mitochondrial respiration and glycolysis of HSPCs.