We acknowledge support by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US Department of Energy (award number DE-FG02- 91ER20021) and National Science Foundation (MCB 0948584) (F.B.). We are thankful to Ms Karen Bird for editing the manuscript.

1. EMS Treatment
Arabidopsis thaliana seeds are mutagenized using as mutagen agent ethyl methane sulfonate (EMS)3,4, which induces into the genome C-to-T changes resulting in C/G to T/A mutations5-7.
<ol>
	<li>Weigh 0.8 g Arabidopsis seeds (~40,000 seeds) carrying the organelle fluorescent marker (specifically, in this study ssGFPHDEL (signal sequence-GFP-HDEL tetrapeptide) has been used as an ER marker).</li>
	<li>Transfer the seeds in a 50-ml falcon tube, then add 25 ml distilled water.</li>
	<li>Add 0.2% (v/v) ethyl methane sulfonate.</li>
	<li>Incubate for 16 hours on nutating mixer at low speed.</li>
	<li>Aspirate the liquid and discard it into a flask containing 1.0 M NaOH to inactivate the EMS.</li>
	<li>Add 25 ml water to the Falcon tube containing the seeds, close, and invert 5 times to wash the seeds; wait until all the seeds have settled, then aspirate the water and discard into the 1.0 M NaOH flask.</li>
	<li>Repeat the washing step up to 10 times.</li>
	<li>After the final wash, re-suspend the seeds in a minimal amount of water.</li>
	<li>Proceed to seed sterilization adding 25 mL of 10 % bleach, shake vigorously for 30 s. Let seeds settle to bottom, then pour off bleach and rinse with 25 mL of sterile water. Pour off the sterile water and add 25 mL 70 % ethanol. Shake tubes for 30 s. Let seeds settle to bottom. Pour off ethanol and rinse with 25 mL of sterile water. Repeat twice the wash with sterile water, then pour seeds on a plastic Petri dish containing 3 MM filter paper and let dry under the hood. Store at 4 &deg;C for 2 days.</li>
	<li>Plate the seeds on &frac12; MS phytagel (half concentration of Murashige and Skoog medium), 150-mm Petri dish (~250-300 seeds for plate).</li>
	<li>Grow M1 seeds on plate for 2 weeks, then transplant into soil.</li>
	<li>Collect M2 seeds from individual M1 plants to generate M2 lines (1000 independent lines).</li>
</ol>
2. Confocal Screening of M2 and M3 Populations
In this section we describe the observation of seedlings with a confocal or fluorescence microscope as described earlier8.
<ol>
	<li>Sixty seeds from each M2 line are grown for 7 to 10 days on plastic Petri dishes, &frac12; MS phytagel; on the same plate are also grown 5 seedlings EMS-untreated (control).</li>
	<li>Five to ten cotyledons are mounted with the abaxial side toward the lens (40X) on a microscope slide and enclosed with a coverslip.</li>
	<li>Each cotyledon is observed, from the cortical to the medial region, under fluorescence for any altered subcellular distribution of the organelle marker.</li>
	<li>Positive plants are transplanted into soil and their seeds are collected and screened again to confirm the mutant phenotype in the M3 generation.</li>
	<li>Remove the contaminating background mutations through backcrossing at least three times to a wild-type genome possibly containing the desired organelle fluorescent marker.
	<ol>
		<li>From the mother plant, using fine scissors or forceps remove mature siliques, and open flowers.</li>
		<li>Remove the buds that are too small from the meristem.</li>
		<li>Insert the tip of one pair of forceps between petals and sepals to open one flower bud and remove all anthers.</li>
		<li>Using one pair of forceps take an open mature flower from the father plant and rub the anthers on the stigma of the emasculated plant.</li>
	</ol>
	</li>
</ol>
3. Mapping
This section describes essentially how to map a recessive mutation using a modified protocol from Borevitz9 which is faster if compared to traditional mapping methods10,11 . This approach use high-density oligonucleotide arrays with the ability to detect numerous single feature polymorphisms (SFPs) in a single assay12. Using the Affymetrix Arabidopsis ATH1 GeneChip array is possible to analyze approximately 24,000 genes. A pool of F2 individuals showing the mutation is compared to a pool of wild-type F2 plants collected within the same segregating population. Then, the mutation will be mapped in the region where the mutant pool is enriched for mutant genotype alleles and consequently in the same region the wild-type pool will result enriched for the wild-type parent alleles13.
<ol>
	<li>Genomic DNA (3 &mu;g) is extracted from the homozygous mutant Columbia (M3) using a Qiagen DNeasy Plant Mini Kit and is submitted for Illumina Genome Analyzer II (GA II)14 sequencing.</li>
	<li>The homozygous mutant Columbia (M3) is crossed with Landsberg erecta to generate a mapping population.</li>
	<li>The mapping is performed on 70 up to 100 F2 individuals showing the aberrant phenotype and the same number of F2 plants with a wild-type phenotype.</li>
	<li>Collect from each plant a leaf disc (0.60 mm) using a hole punch. The leaf disc should be collected from leaves of the same age to be sure to have similar amounts of DNA. The samples can be processed for genomic extraction separately or in groups. In this case, it is possible to collect 5-10 samples for each Eppendorf tube so that in the end you will have fewer eppendorf tubes from which genomic DNA has to be extracted.</li>
	<li>MasterPure Plant Leaf DNA Purification Kit (Epicentre) is used to extract the genomic DNA. The genomic DNA obtained from each sample is quantified with a nanodrop.</li>
	<li>The same quantity of genomic DNA from each sample is then put together up to a total of 300 ng (~30 &mu;L) of plant DNAs; add 60 &mu;L 2.5X random primers solution [125 mM Tris-HCl (pH 6.8), 12.5 mM MgCl2, 25 mM 2-mercaptoethanol, 750 &mu;g/ml oligodeoxyribonucleotide primers (random octamers)] (Bioprime kit) and 42 &mu;L water (final volume 132 &mu;L).</li>
	<li>Denature DNAs at &gt;95 &deg;C for 5 to 10 min.</li>
	<li>Cool on ice.</li>
	<li>To each denatured DNA add 15 &mu;L 10X dNTPs mix with Biotin dCTP [1 mM biotin-14-dCTP, 1 mM dCTP, 2 mM dATP, 2 mM dGTP, 2 mM dTTP in 10 mM Tris-HCl (pH 7.5), 1 mM Na2EDTA] (Bioprime kit), and complete with 3 &mu;L Klenow polymerase (Bioprime kit).</li>
	<li>Incubate overnight at 25 &deg;C.</li>
	<li>On the following day, add 15 &mu;L 3 M NaOAc and 400 &mu;L cold 100% EtOH; mix.</li>
	<li>Incubate at -80 &deg;C for 1 hour, then spin at 20,500x g for 15 min, remove supernatant, and wash with 500 &mu;L cold 75% EtOH.</li>
	<li>Spin at 20,500x g for 10 min.</li>
	<li>Dry DNA pellets at 37 &deg;C for 10 min and resuspend in 100 &mu;L water.</li>
	<li>Use 5 &mu;L to check yield and quality on a gel (Figure 2).</li>
	<li>Send 95 &mu;L of labeling reaction for the wild-type and mutant for GeneChip Arabidopsis ATH1 Genome Array Hybridization.</li>
	<li>The arrays are scanned and the .CEL files obtained are analyzed using R software (<a href="http://www.r-project.org/">http://www.r-project.org/</a>).</li>
	<li>Install R software, then open the program and paste the following string: 
	source("<a href="http://bioconductor.org/biocLite.R">http://bioconductor.org/biocLite.R</a>") 
	biocLite() 
	Then press return key-this will install the standard Bioconductor packages (<a href="http://www.bioconductor.org">http://www.bioconductor.org</a>).</li>
	<li>In a new folder on your desktop, download from the following website (<a href="http://www.naturalvariation.org/methods">http://www.naturalvariation.org/methods</a>) the files: 
	 readcel.R 
	 SFP.R 
	 Map.R 
	 ath1V5.RData 
	 ColLerCEL.zip 
	The file ColLerCEL.zip should be unpacked and the resulting files pasted into your new folder.</li>
	<li>Rename the data obtained from your GeneChip experiment into wildtype.CEL and mutant.CEL.</li>
	<li>Copy now your GeneChip data (wildtype.CEL and mutant.CEL) into your folder.</li>
	<li>Open R, a) for Mac: click "Misc," and then click "Change working directory." b) for PC: click "File" then "Change directory"</li>
	<li>a) for Mac: Select the folder containing the files. Click "Workspace," "Load workspace file" and select ath1V5.RData, b) for PC: Select the folder containing the files above, click "File," then "Load workspace" and select ath1V5.RData.</li>
	<li>Open readCEL.R using Notepad and copy the whole text to R.</li>
	<li>Open SFP.R using Notepad and copy the whole text to R.</li>
	<li>Open Map.R using Notepad and copy the whole text to R.</li>
	<li>In the Console window you will see the following message: 
	Chromosome x limits Mb y z 
	Search genes at TAIR paste in this link 
	3<a href="http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z">http://arabidopsis.org/servlets/sv?action=download&amp;chr=x&amp;start=y&amp;end=z</a></li>
	<li>Copy and paste the link into your internet browser. A window will appear and ask to open or save the file; you should select save. Choose a file name and attach the extension ".xls," then click Save.</li>
	<li>Open the file ".xls" where you can find the coordinate for your mutant (the result is graphically represented in Figure 3).</li>
	<li>Within the mapped area in the assembled Illumina reads of the mutant genome identify specific EMS transitions 4 among the single nucleotide polymorphisms. Complement the mutant phenotype by transformation with wild type gene(s) using standard procedures15.</li>
</ol>
4. Representative Results
Figure 1 shows the approach used for identification of a mutant of the secretory pathway using confocal microscopy screening. Figure 2 shows a typical preparation of labeled genomic DNA for array hybridization. In figure 3, a typical result expected after analyzing the data obtained from the GeneChip Arabidopsis ATH1 Genome Array is presented.
<img alt="Figure 1" src="/files/ftp_upload/3809/3809fig1.jpg" /> 
Figure 1. Arabidopsis transgenic plants expressing ssGFPHDEL (ER marker) (1) were cultivated to produce enough seeds for EMS treatment (2). Seeds treated with EMS were then sown to generate M1 plants (3). Each M1 plant represents a different line and seed were collected separately from each of them. M2 seed were plated on &frac12; MS substrate (4) and then screened for defects in ER morphology by confocal microscopy (5). During the screening we found plants that conserved the wild-type ER morphology (6) and plants that show defective ER phenotypes (7). These plants were grown to obtain the M3 generation and to confirm the phenotype (8). Genomic DNA from M3 plant was used for Solexa Illumina sequencing (a). The same plant was also used for crosses with Ler-wt to obtain the F2 mapping population (b).
<img alt="Figure 2" src="/files/ftp_upload/3809/3809fig2.jpg" /> 
Figure 2. Bioprime random labeling reactions (5 &mu;L of 100 &mu;L) were loaded on agarose gel 1%. Lane a is from a pool of wild-type F2 plants, and lane b is from a pool of mutant F2 plants. (Marker is 1 Kb DNA ladder-N3232, from NEB).
<img alt="Figure 3" src="/files/ftp_upload/3809/3809fig3.jpg" /> 
Figure 3. The figure represents an example of mapping of Col-0 mutation using GeneChip Arabidopsis ATH1 Genome Array Hybridization. The mutation in this example is located on chromosome 1, delimited by vertical bars. The horizontal bars represent the thresholds for detection.

<ol>
	<li>Marti, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+missense+mutation+in+the+vacuolar+protein+GOLD36+causes+organizational+defects+in+the+ER+and+aberrant+protein+trafficking+in+the+plant+secretory+pathway.">A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway.</a> The Plant journal : for cell and molecular biology. 63, 901-913 (2010).</li><li>Stefano, G., Renna, L., Moss, T., McNew, J., Brandizzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+the+spatial+and+dynamic+organization+of+the+endoplasmic+reticulum+and+Golgi+apparatus+is+influenced+by+the+integrity+of+the+c-terminal+domain+of+RHD3,+a+non-essential+GTPase.">Arabidopsis the spatial and dynamic organization of the endoplasmic reticulum and Golgi apparatus is influenced by the integrity of the c-terminal domain of RHD3, a non-essential GTPase.</a> The Plant Journal. , (2011).</li><li>Kim, Y., Schumaker, K. S., Zhu, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EMS+mutagenesis+of+Arabidopsis.">EMS mutagenesis of Arabidopsis.</a> Methods in molecular biology. 323, 101-103 (2006).</li><li>Maple, J., Moller, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutagenesis+in+Arabidopsis.">Mutagenesis in Arabidopsis.</a> Methods in molecular biology. 362, 197-206 (2007).</li><li>Greene, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectrum+of+chemically+induced+mutations+from+a+large-scale+reverse-genetic+screen+in+Arabidopsis.">Spectrum of chemically induced mutations from a large-scale reverse-genetic screen in Arabidopsis.</a> Genetics. 164, 731-740 (2003).</li><li>Krieg, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethyl+methanesulfonate-induced+reversion+of+bacteriophage+T4rII+mutants.">Ethyl methanesulfonate-induced reversion of bacteriophage T4rII mutants.</a> Genetics. 48, 561-580 (1963).</li><li>Kovalchuk, I., Kovalchuk, O., Hohn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+variation+of+the+somatic+mutation+frequency+in+transgenic+plants.">Genome-wide variation of the somatic mutation frequency in transgenic plants.</a> The EMBO journal. 19, 4431-4438 (2000).</li><li>Boulaflous, A., Faso, C., Brandizzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+the+Golgi+apparatus:+from+imaging+to+genes.">Deciphering the Golgi apparatus: from imaging to genes.</a> Traffic. 9, 1613-1617 (2008).</li><li>Borevitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotyping+and+mapping+with+high-density+oligonucleotide+arrays.">Genotyping and mapping with high-density oligonucleotide arrays.</a> Methods in molecular biology. 323, 137-145 (2006).</li><li>Konieczny, A., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+mapping+Arabidopsis+mutations+using+co-dominant+ecotype-specific+PCR-based+markers.">A procedure for mapping Arabidopsis mutations using co-dominant ecotype-specific PCR-based markers.</a> The Plant journal : for cell and molecular biology. 4, 403-410 (1993).</li><li>Bell, C. J., Ecker, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assignment+of+30+microsatellite+loci+to+the+linkage+map+of+Arabidopsis.">Assignment of 30 microsatellite loci to the linkage map of Arabidopsis.</a> Genomics. 19, 137-144 (1994).</li><li>Hazen, S. P., Kay, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+arrays+are+not+just+for+measuring+gene+expression.">Gene arrays are not just for measuring gene expression.</a> Trends in Plant Science. 8, 413-416 (2003).</li><li>Hazen, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+array+mapping+of+circadian+clock+and+developmental+mutations+in+Arabidopsis.">Rapid array mapping of circadian clock and developmental mutations in Arabidopsis.</a> Plant Physiology. 138, 990-997 (2005).</li><li>Bentley, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+whole+human+genome+sequencing+using+reversible+terminator+chemistry.">Accurate whole human genome sequencing using reversible terminator chemistry.</a> Nature. 456, 53-59 (2008).</li><li>Weigel, D., Glazebrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis.">Arabidopsis.</a> A Laboratory Manual. , (2002).</li><li>Voelkerding, K. V., Dames, S., Durtschi, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next+generation+sequencing+for+clinical+diagnostics-principles+and+application+to+targeted+resequencing+for+hypertrophic+cardiomyopathy:+a+paper+from+the+2009+William+Beaumont+Hospital+Symposium+on+Molecular+Pathology.">Next generation sequencing for clinical diagnostics-principles and application to targeted resequencing for hypertrophic cardiomyopathy: a paper from the 2009 William Beaumont Hospital Symposium on Molecular Pathology.</a> J. Mol. Diagn. 12, 539-551 (2010).</li></ol>

We have nothing to disclose.

Here we described a confocal microscopy-based screening for the identification of endomembrane mutants. This approach can be easily extended to other organelles of the cell for which specific fluorescent protein markers are available. The screen is based on the identification of mutants that show an aberrant distribution of the fluorescent marker either in the target organelle or to organelles that are not supposed to contain the marker. Respectively, these mutants represent populations in which either the ability of the...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Ethylmethane sulfonate</td>
 <td>Sigma</td>
 <td>M0880</td>
 </tr>
 <tr>
 <td>NaOH</td>
 <td>J.T Baker</td>
 <td>3722-05</td>
 </tr>
 <tr>
 <td>Murashige skoog basal medium w gamborg vitamins</td>
 <td>Phyto technolog laboratorie</td>
 <td>M404</td>
 </tr>
 <tr>
 <td>Phytagel</td>
 <td>Sigma</td>
 <td>P8169-1Kg</td>
 </tr>
 <tr>
 <td>RNeasy mini kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>Master pure plant leaf DNA purification kit</td>
 <td>Epicentre</td>
 <td>MPP92100</td>
 </tr>
 <tr>
 <td>Bioprime DNA labeling system</td>
 <td>Invitrogen</td>
 <td>18094-011</td>
 </tr>
 <tr>
 <td>Alcohol 200 proof</td>
 <td>Decan laboratories inc.</td>
 <td>2716</td>
 </tr>
 <tr>
 <td>NaOAc</td>
 <td>J.T Baker</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Gene chip Arabidopsis ATH1 genome array</td>
 <td>Affymetrix</td>
 <td>900385</td>
 </tr>
 <tr>
 <td>Falcon tubes 50 mL</td>
 <td>corning</td>
 <td>430290</td>
 </tr>
 <tr>
 <td>Eppendorf tubes 1.5 mL</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Filter paper 90mm</td>
 <td>Whatman</td>
 <td>1001090</td>
 </tr>
 <tr>
 <td>Analytical Balance</td>
 <td>Mettler Toledo AB54-S</td>
 <td>n.a</td>
 </tr>
 <tr>
 <td>Nutating (wave) shaker</td>
 <td>Heidolph polymax 1040</td>
 <td>n.a</td>
 </tr>
 <tr>
 <td>Centrifuge</td>
 <td>Eppendorf 5417-R</td>
 <td>n.a</td>
 </tr>
 </tbody>
</table>

fluorescence-microscopy screening and next-generation sequencing: useful tools for the identification of genes involved in organelle integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

Watch this Scientific Journal Video about Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity at JoVE.com

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

fluorescence-microscopy-screening-next-generation-sequencing-useful

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Biology

12.2K 次观看.  Michigan State University. 在细胞生物学的一个基本任务是定义机制的基础，使真核细胞的细胞器的身份。在这里，我们提出了一个方法，以确定该基因的植物细胞器，利用荧光显微镜和下一代测序工具的形态和功能的完整性负责。

视频: Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...