This work was supported by the National Key Research and Development Program of China (No. 2016YFD0102103) and the National Natural Science Foundation of China (No.31701394).

1. Preparations
<ol>
	<li>Development of &#947;-rays mutagenized populations
	<ol>
		<li>Treat about 20,000 dried rice seeds (with moisture content of ca. 14%) of a japonica rice line (e.g., DS552) with 137Cs gamma rays at 100 Gy (1 Gy/min) in a &#947; irradiation facility (e.g., gamma cell). 
		NOTE: Seeds used for treatment should have a high viability (e.g., with a germination rate &#62;85%). The irradiation dose for indica rice could be increased to 150 Gy.</li>
		<li>Sow the irradiated seeds after germination on a seedling bed and transplant seedlings individually to a paddy field and grow into the M1 population. 
		NOTE: Direct seeding of M1 plants sparsely could also be applied to save labor cost. Prevent outcrossing of M1 plants with other rice varieties using physical or biological isolation means.</li>
		<li>Bulk-harvest M2 seeds from the M1 plants, with 1-2 seeds from each M1 panic, to form an M2 population. 
		NOTE: In practice and for simplicity, harvest all seeds of M1 plants and after fully mixing, a portion is sampled to form an M2 population.</li>
		<li>Soak about 5,000 M2 seeds in water for 24 (indica rice)-36 (japonica rice) h at room temperature. Then let the seeds to germinate at 37 &#176;C for 2 days on moist filter paper in Petri dishes. 
		NOTE: More M2 seeds can be germinated for analysis to increase the probability of identifying mutants.</li>
		<li>Place the germinated seeds to seeding panels with small holes and grow them hydroponically for 3-4 weeks in a culture solution modified from Yoshida et al.15 in a glasshouse with a 12 h photoperiod [daytime (30&#177;2) &#176;C and night (24&#177;2) &#176;C].</li>
	</ol>
	</li>
	<li>Sampling of leaf tissues: Cut one disk (&#934; ~2 mm) from the fully extended leaf of each seeding at the same position using a hole puncher.</li>
	<li>Preparation of DNA extraction solutions
	<ol>
		<li>Buffer A: Add 2 mL of 5 M NaOH and 10 mL of 20% Tween 20 to make the final volume of 50 mL. Freshly prepare buffer A before DNA extraction.</li>
		<li>Buffer B: Add 20 mL of 1 M Tris-HCl (pH 8.0) and 80 &#956;L of 0.5 M EDTA to make a final volume of 100 mL.</li>
	</ol>
	</li>
	<li>PCR primers
	<ol>
		<li>Primers for HRM analysis: Design primers for amplification of the target sequence using software (e.g., Primer Premier5) and synthesize by a commercial company. 
		NOTE: Because HRM is less applicable to the analysis of long fragments, and hence, the amplicons should be less than 400 bp. Fragments with too high (&#62;75%) or too low (&#60;25%) GC content are also not good for HRM analysis.</li>
		<li>Primers for quality control: Use the 24 SSR markers distributed on the 12 rice chromosomes from Peng et al.16.</li>
	</ol>
	</li>
</ol>
2. DNA Extraction
<ol>
	<li>Place 4 leaf discs into each well of a 96-well PCR plate, add buffer A solution (50 mL/well). Freeze the plate in a -80 &#176;C freezer for 10 min.</li>
	<li>Defreeze the plate at room temperature, and then incubate at 95 &#176;C for 10 min.</li>
	<li>Add 50 &#956;L of buffer B to each well and mix well by vortexing.</li>
	<li>Centrifuge the plate for 1 min at 1,500 x g. The supernatant is ready for PCR.</li>
</ol>
3. PCR Amplification
<ol>
	<li>PCR Optimization
	<ol>
		<li>Add the following reagents to each PCR well: 1 &#956;L of DNA (the supernatant in step 2.4), 5 &#956;L of 2x Master Mix (containing 2x PCR buffer, 4 mmol/L MgCl2, 0.4 mmol/L 2&#39;-deoxyribonucleoside triphosphates (dNTPs), and 50 U/mL Taq DNA polymerase, 0.2 &#956;L each of 10 &#956;mol/L primers, and make a final volume up to 10 &#956;L using nuclease free water.</li>
		<li>Use a gradient-capable thermal block to determine the optimal annealing temperature for each target fragment by using the following PCR program: 5 min at 94 &#176;C, followed by 40 cycles of 30 s at 94 &#176;C, 30 s at 52-62 &#176;C (gradient temperature), and 30 s at 72 &#176;C, with a final extension at 72 &#176;C for 8 min and a hold at 16 &#176;C.</li>
		<li>Examine amplicons on 1% agarose gels for determination of the optimal annealing temperature. 
		NOTE: An optimal annealing temperature should enable specific amplification of the target fragment, without nonspecific amplification and primer dimerization.</li>
	</ol>
	</li>
	<li>PCR for HRM analysis 
	NOTE: HRM compatible plates and DNA of M2 seedlings extracted as described in step 2.4 are used for PCR.
	<ol>
		<li>Perform PCRs in a final volume of 10 &#956;L, with 1 &#956;L of DNA (supernatant), 5 &#956;L of 2x Master Mix, 0.2 &#956;L each of 10 &#956;mol/L primers, and 1 &#956;L of 10x fluorescence dye. In each plate include one wild type (WT) parented sample and one negative (without DNA) control.</li>
		<li>Add a drop of mineral oil to each well to prevent evaporation.</li>
		<li>Seal the plate with adhesive film and centrifuge at 1,000 x g for 1 min.</li>
		<li>Run the PCR using the optimized annealing temperature. 
		NOTE: In a few cases, fluorescence dye may affect PCR amplification, hence the dye is added after completion of PCR. In such cases, the dye is incorporated into DNA strands by post-PCR denaturing and annealing.</li>
	</ol>
	</li>
</ol>
4. HRM Scanning and Mutation Confirmation
<ol>
	<li>Remove the adhesive film from plate and insert the plate into an HRM machine.</li>
	<li>Select New Run from the file menu or press the Run button at the top of the screen.</li>
	<li>Specify the starting and ending temperatures for the melt from 55 &#176;C to 95 &#176;C. 
	NOTE: After the first run, the range of melt temperature can be determined for a particular fragment; hence, the melt temperature can be adjusted for subsequent analysis of the same fragment to save time.</li>
	<li>Select samples for high resolution melting analysis, exclude samples similar to the negative control.</li>
	<li>Normalize the melting curves to have the same beginning and ending fluorescence. Visually confirm that the Lower Min and Lower Max temperature cursors are in a region of the curves.</li>
	<li>Keep the &#8710;F (difference of fluorescence) level at the default setting of 0.05.</li>
	<li>Select the Common versus Variant from the Standards selection list and choose the Normal sensitivity.</li>
	<li>Use the WT as the control; samples with a &#8710;F value of &#8805;0.05 from WT are considered to contain mutant plant.</li>
	<li>Identification and confirmation of mutant plants.
	<ol>
		<li>Identify the four plants, of which each mutant pool was made.</li>
		<li>Extract DNA from each of these plants using a CTAB method according to Allen et al.17 with some modifications. 
		NOTE: DNase-free RNase and NaAc are not used when extracting DNA using the CTAB method described by Allen et al.17, as the DNA quality is good enough for further PCR amplification. Adjust the DNA to a final concentration of ~25 ng/&#956;L after quantification using a spectrophotometer.</li>
		<li>Amplify the target fragment using PCR primers and program the same as for HRM analysis.</li>
		<li>Identify specific molecular lesions by Sanger sequencing of the amplicons. 
		NOTE: Once the completion of PCR amplification, send the amplicons to a company for Sanger sequencing. The molecular lesion can be identified by comparing the sequences between the M2 plant and the WT.</li>
	</ol>
	</li>
</ol>
5. Quality Control of Selected Mutants with Molecular Markers
<ol>
	<li>Perform PCR for the 24 SSR markers in a final volume of 10 &#956;L with 25 ng of genomic DNA (extracted using the CTAB protocol), 5 &#181;L of 2x master mix, 0.2 &#181;L of each of 10 &#956;M SSR primers.</li>
	<li>Run PCR using the following program: 5 min at 94 &#176;C, followed by 30 cycles of 30 s at 94 &#176;C, 30 s at 55 &#176;C and 30 s at 72 &#176;C, with a final extension at 72 &#176;C for 7 min.</li>
	<li>Separate the amplicons on 8% polyacrylamide gels and reveal polymorphism of amplified fragments by silver staining18.</li>
	<li>Compare the SSR haplotypes of selected variants and the WT. 
	NOTE: Induced mutants often have SSR haplotypes identical to their WT, if more than one SSR markers are different between a variant and the WT, the variant is high likely to be a genetic contaminant (e.g., a mixture or outcrossed plant) rather than an induced mutant18.</li>
</ol>

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M., Krysan, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ITILLING:+a+personalized+approach+to+the+identification+of+induced+mutations+in+arabidopsis.">ITILLING: a personalized approach to the identification of induced mutations in arabidopsis.</a> Physiology. 154 (1), 25-35 (2010).</li><li>Colasuonno, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DHPLC+technology+for+high-throughput+detection+of+mutations+in+a+durum+wheat+TILLING+population.">DHPLC technology for high-throughput detection of mutations in a durum wheat TILLING population.</a> BMC Genetics. 17 (1), 43 (2016).</li><li>Tsai, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+of+rare+mutations+in+populations:+TILLING+by+sequencing.">Discovery of rare mutations in populations: TILLING by sequencing.</a> Plant Physiology. 156, 1257-1268 (2011).</li><li>Kumar, A. 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The authors declare that they have no conflict of interest.

TILLING has proved to be a powerful reverse genetic tool for identifying induced mutations for gene functional analysis and crop breeding. For some traits not easily observed or determined, TILLING with high-throughput PCR-based mutation detection can be a useful method to obtain mutants for different genes. HRM-TILLING method has been used in EMS-mutagenized populations of tomato12, wheat11 and grapevine20 for mutation screening. In this paper, a si...

Mutants are important genetic resources for plant functional genomics research and breeding of new varieties. A forward genetics approach (i.e. from mutant selection to gene cloning or variety development) used to be the main and sole method for the use of induced mutations about 20 years ago. The development of a novel reverse genetics method, TILLING (Targeting Induced Local Lesions In Genomes) by McCallum et al.1 opened a new paradigm and it has since been applied in a great number of animal and plant species2. TILLING is particularly useful for breeding traits that are technically difficult or costly to be determined (e.g., disease resistance, mineral content).
TILLING was initially developed for screening point mutations induced by chemical mutagens (e.g., EMS1,3). It includes the following steps: the establishment of a TILLING population(s); DNA preparation and pooling of individual plants; PCR amplification of target DNA fragment; heteroduplexes formation by denaturation and annealing of PCR amplicons and cleavage by CEL I nuclease; and identification of mutant individuals and their specific molecular lesions3,4. However, this method is still relatively complex, time consuming, and low-throughput. To make it more efficient and with higher throughput, many modified TILLING methods have been developed, such as deletion TILLING (De-TILLING) (Table 1)1,3,5,6,7,8,9,10,11,12.
HRM curve analysis, which is based on fluorescence changes during the melting of the DNA duplex, is a simple, cost-effective, and high-throughput method for mutation screening and genotyping13. HRM has already been widely used in plant research including HRM based TILLING (HRM-TILLING) for screening SBS mutations induced by EMS mutagenesis14. Here, we presented detailed HRM-TILLING protocols for screening of mutations (both Indel and SBS) induced by gamma (&#947;) rays in rice.

<table>
	<tbody>
		<tr>
			<td>2&#215; Taq plus PCR Master Mix</td>
			<td>Tiangen, China</td>
			<td>KT201</td>
			<td>PCR buffer, dNTP and polymerase for PCR amplification</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Bio-rad, America</td>
			<td>MSP-9651</td>
			<td>Specific plate for PCR in HRM analysis</td>
		</tr>
		<tr>
			<td>Mastercycler nexus</td>
			<td>Eppendorf, German</td>
			<td>6333000073</td>
			<td>PCR amplification</td>
		</tr>
		<tr>
			<td>LightScanner</td>
			<td>Idaho Technology, USA</td>
			<td>LCSN-ASY-0011</td>
			<td>For fluorescence sampling and processing</td>
		</tr>
		<tr>
			<td>CALL-IT 2.0</td>
			<td>Idaho Technology, USA</td>
			<td></td>
			<td>For analysis of the fluorescence change</td>
		</tr>
		<tr>
			<td>EvaGreen</td>
			<td>Biotium, USA</td>
			<td>31000-T</td>
			<td>Fluorescence dye of HRM</td>
		</tr>
		<tr>
			<td>Nanodrop 2000</td>
			<td>Thermo Scientific, USA</td>
			<td>ND2000</td>
			<td>For DNA quantification</td>
		</tr>
	</tbody>
</table>

identifying mutations by high resolution melting in a tilling population of rice

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the TILLING system has less applicability for insertion/deletion (Indel) detection and traditional TILLING needs more complex steps, like CEL I nuclease digestion and gel electrophoresis. To improve the throughput and selection efficiency, and to make the screening of both Indels and single base substitions (SBSs) possible, a new high-resolution melting (HRM)-based TILLING system is developed. Here, we present a detailed HRM-TILLING protocol and show its application in mutation screening. This method can analyze the mutations of PCR amplicons by measuring the denaturation of double-stranded DNA at high temperatures. HRM analysis is directly performed post-PCR without additional processing. Moreover, a simple, safe and fast (SSF) DNA extraction method is integrated with HRM-TILLING to identify both Indels and SBSs. Its simplicity, robustness and high throughput make it potentially useful for mutation scanning in rice and other crops.

HRM Scanning and Analysis
In total, 1,140 pooled DNA samples from 4,560 M2 seedlings were produced and subjected to PCR amplification. Two fragments with the size of 195 bp and 259 bp were amplified for OsLCT1 and SPDT, respectively (Table 2). Most samples had melting curves not significantly different from the WT (&#916;F &#60; 0.05). HRM curves significantly different from the WT (&#916;F &#62; 0.05) were grouped with color(s) di...

Watch this Scientific Journal Video about Identifying Mutations by High Resolution Melting in a TILLING Population of Rice at JoVE.com

Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

Target Induced Local Lesions In Genomes (TILLING) is a strategy of reverse genetics for the high-throughput screening of induced mutations. However, the ...

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7.3K Views.  Zhejiang University. In this article, we present the protocol that is described as high-resolution melting analysis (HRM)-based Target Induced Local Lesions In Genomes (TILLING). This method utilizes fluorescence changes during the melting of the DNA duplex and is suitable for high-throughput screening of both insertion/deletion (Indel) and single base substition (SBS).

Video: Identifying Mutations by High Resolution Melting in a TILLING Population of Rice

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer's Disease Gene Susceptibility Assessment

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but especially on red blood cells (RBCs). As a receptor for the complement components C3b and C4b, CR1 regulates the activation of the complement cascade and promotes the phagocytosis of immune complexes and cellular debris, as well as the amyloid-beta (A&#946;) peptide in Alzheimer's disease (AD). Several studies have confirmed AD-associated single nucleotide polymorphisms (SNPs), as well as a copy-number variation (CNV) in the CR1 gene. Here, we describe an innovative method for determining the length polymorphism of the CR1 receptor. The receptor includes three domains, called long homologous repeats (LHR)-LHR-A, LHR-C, and LHR-D-and an n domain, LHR-B, where n is an integer between 0 and 3. Using a single pair of specific primers, the genetic material is used to amplify a first fragment of the LHR-B domain (the variant amplicon B) and a second fragment of the LHR-C domain (the invariant amplicon). The variant amplicon B and the invariant amplicon display differences at five nucleotides outside of the hybridization areas of said primers. The numbers of variant amplicons B and of invariant amplicons is deduced using a quantitative tool (high-resolution melting (HRM) curves), and the ratio of the variant amplicon B to the invariant amplicon differs according to the CR1 length polymorphism. This method provides several advantages over the canonical phenotype method, as it does not require fresh material and is cheaper, faster, and therefore applicable to larger populations. Thus, the use of this method should be helpful to better understand the role of CR1 isoforms in the pathogenesis of diseases such as AD.

High-resolution Melting PCR for Complement Receptor 1 Length Polymorphism Genotyping: An Innovative Tool for Alzheimer&#039;s Disease Gene Susceptibility Assessment

Here, we describe an innovative method to determine complement receptor 1 (CR1) length polymorphisms for use in several applications, particularly the assessment of susceptibility to diseases such as Alzheimer&#39;s disease (AD). This method could be useful to better understand the role of CR1 isoforms in the pathogenesis of AD.

Complement receptor 1 (CR1), a transmembrane glycoprotein that plays a key role in the innate immune system, is expressed on many cell types, but ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms remain elusive mainly due to the lack of a good in vivo model for studying BEST1 and its mutations under physiological conditions. BEST1 encodes an ion channel, namely BESTROPHIN1 (BEST1), which functions in retinal pigment epithelium (RPE); however, the extremely limited accessibility to native human RPE cells represents a major challenge for scientific research. This protocol describes how to generate human RPEs bearing BEST1 disease-causing mutations by induced differentiation from human pluripotent stem cells (hPSCs). As hPSCs are self-renewable, this approach allows researchers to have a steady source of hPSC-RPEs for various experimental analyses, such as immunoblotting, immunofluorescence, and patch clamp, and thus provides a very powerful disease-in-a-dish model for BEST1-associated retinal conditions. Notably, this strategy can be applied to study RPE (patho)physiology and other genes of interest natively expressed in RPE.

Differentiation, Maintenance, and Analysis of Human Retinal Pigment Epithelium Cells: A Disease-in-a-dish Model for BEST1 Mutations

Here we present a protocol to differentiate retinal pigment epithelium (RPE) cells from human pluripotent stem cells bearing patient-derived mutations. The mutant cell lines may be used for functional analyses including immunoblotting, immunofluorescence, and patch clamp. This disease-in-a-dish approach circumvents the difficulty of obtaining native human RPE cells.

Although over 200 genetic mutations in the human BEST1 gene have been identified and linked to retinal degenerative diseases, the pathological mechanisms ...

High-Resolution Comparison of Bacterial Conjugation Frequencies

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons of conjugation frequency under different conditions are required to understand how the conjugative element spreads in nature. However, conventional methods for comparing conjugation frequency are not appropriate for in-depth comparisons because of the high background caused by the occurrence of additional conjugation events on the selective plate. We successfully reduced the background by introducing a most probable number (MPN) method and a higher concentration of antibiotics to prevent further conjugation in selective liquid medium. In addition, we developed a protocol for estimating the probability of how often donor cells initiate conjugation by sorting single donor cells into recipient pools by fluorescence-activated cell sorting (FACS). Using two plasmids, pBP136 and pCAR1, the differences in conjugation frequency in Pseudomonas putida cells could be detected in liquid medium at different stirring rates. The frequencies of conjugation initiation were higher for pBP136 than for pCAR1. Using these results, we can better understand the conjugation features in these two plasmids.

With an aim to understand the behaviors of various bacterial conjugative DNA elements under different conditions, we describe a protocol for detecting differences in conjugation frequency, with high resolution, to estimate how efficiently the donor bacterium initiates conjugation.

Bacterial conjugation is an important step in the horizontal transfer of antibiotic resistance genes via a conjugative DNA element. In-depth comparisons ...

Designing Automated, High-throughput, Continuous Cell Growth Experiments Using eVOLVER

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring fitness phenotypes and improving our understanding of how genotypes are shaped by selection. Extensive recent efforts to develop and apply niche continuous culture devices have revealed the benefits of conducting new forms of cell culture control. This includes defining custom selection pressures and increasing throughput for studies ranging from long-term experimental evolution to genome-wide library selections and synthetic gene circuit characterization. The eVOLVER platform was recently developed to meet this growing demand: a continuous culture platform with a high degree of scalability, flexibility, and automation. eVOLVER provides a single standardizing platform that can be (re)-configured and scaled with minimal effort to perform many different types of high-throughput or multi-dimensional growth selection experiments. Here, a protocol is presented to provide users of the eVOLVER framework a description for configuring the system to conduct a custom, large-scale continuous growth experiment. Specifically, the protocol guides users on how to program the system to multiplex two selection pressures &#8212; temperature and osmolarity &#8212; across many eVOLVER vials in order to quantify fitness landscapes of Saccharomyces cerevisiae mutants at fine resolution. We show how the device can be configured both programmatically, through its open-source web-based software, and physically, by arranging fluidic and hardware layouts. The process of physically setting up the device, programming the culture routine, monitoring and interacting with the experiment in real-time over the internet, sampling vials for subsequent offline analysis, and post experiment data analysis are detailed. This should serve as a starting point for researchers across diverse disciplines to apply eVOLVER in the design of their own complex and high-throughput cell growth experiments to study and manipulate biological systems.

The eVOLVER framework enables high-throughput continuous microbial culture with high resolution and dynamic control over experimental parameters. This protocol demonstrates how to apply the system to conduct a complex fitness experiment, guiding users on programming automated control over many individual cultures, measuring, collecting, and interacting with experimental data in real-time.

Continuous culture methods enable cells to be grown under quantitatively controlled environmental conditions, and are thus broadly useful for measuring ...

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.