CUSTOM SERVICES · 2019-01-22 · services for whole transcriptome sequencing and gene expression...
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CUSTOM SERVICES for epigenetics and gene regulation research • ChIP-Seq • DNA Methylation • Histone Analysis • Gene Regulation • miRNA
Transcript of CUSTOM SERVICES · 2019-01-22 · services for whole transcriptome sequencing and gene expression...
Enabling Epigenetics Research
CUSTOM SERVICES for epigenetics and gene regulation research
• ChIP-Seq• DNA Methylation• Histone Analysis• Gene Regulation• miRNA
CUSTOM SERVICES for epigenetics and gene regulation research
• ChIP Antibody Validation
• RNA-Seq
• DNA Methylation
• Interactome Profiling
• miRNA Functional Analysis
• Cell-based Pathway Analysis
• Multiplex Histone Modification Analysis
• Reporter Assay Screens
The Active Motif Custom Services team makes cutting-edge research accessible to the wider life science community. We provide services for state-of-the-art epigenetics and gene regulation analysis techniques to accelerate your research.
For a complete list of available products, please visit us at www.activemotif.com.
• ChIP-Seq
ChIP-SEQend-to-end services for genome-wide mapping of protein-DNA interactions
Chromatin immunoprecipitation combined with Next-Generation Sequencing (ChIP-Seq) is the most widely utilized technique to study protein-DNA interactions and histone modification localization across the genome. Given the importance of ChIP-Seq data sets for development and disease research, obtaining the highest quality data is crucial.
CHOOSE THE GLOBAL LEADER IN END-TO-END ChIP SERVICESActive Motif offers the most diversified portfolio for ChIP Services. We bring over a decade of experience providing services, with over 10,000 samples processed, and the highest level of expertise of any service provider.
ChIP SERVICES| ChIP-Seq
map protein-DNA interactions and histone modifications
| Low Cell Number ChIP-Seqstarting from as few as 10,000 cells
| ChIP Antibody Validationverify that your antibody works in ChIP
| Super-enhancer Profilingchoose from our validated super-enhancer targets
| ChIP-Seq Spike-ina novel normalization strategy
| RNA Pol II ChIP-Seqmeasure transcription rates
| ChIP-qPCRcustom analysis of any gene
For more information and a complete list of available genome-wide services, please visit us at www.activemotif.com/services.
Chromatin preparation is quantified and sized
Antibody is ChIP Qualified prior to ChIP-Seq ChIP rxns
qPCR is used to validate success of the ChIP rxns
QC by quantification of yield and library size
Sequencing to yield≥ 30 million tags
Customer fixes cell lines or freezes tissue samples
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Active Motif prepares chromatin and sonicates
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Active Motif performs the ChIP reactions
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Active Motif constructsChIP-Seq libraries
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Libraries sequenced usingIllumina platform
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Analysis and Data Delivery6
Comprehensive ChIP-Seq Services
Quality Control Stepsto Ensure Success
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END-TO-END LOW CELL NUMBER CHIP-SEQ SERVICESOne of the major obstacles preventing researchers from generating valuable ChIP-Seq data sets is the limited cell numbers available for many cell systems. Performing ChIP-Seq using small quantities of starting materials is difficult when compared to standard ChIP-Seq experiments that start with 10 million cells. Advances in Active Motif’s ChIP-Seq services protocol have resolved issues often associated with low-cell ChIP, including poor signal-to-noise in the ChIP reactions, inefficient library amplification and high duplication rates in the final ChIP-Seq data sets.
Active Motif’s Services group has adapted and optimized its ChIP-Seq protocols and pipeline to enable the generation of high quality ChIP-Seq data from as few as 10,000 cells. We offer Low Cell Number ChIP-Seq as an end-to-end service. Just submit your cell pellets or small frozen tissue samples to us and we’ll do the rest.
LOW CELL ChIP-SEQ FEATURES
• Get data from as few as 10,000 cells
• Histone marks or transcription factors
• Sorted cells or small tissue biopsies
• Cells to data in 8-10 weeks
Standard ChIP-Seq protocols require 10 million cells to perform ChIP-Seq, a number that is often unattainable for many cell types. Advances in Active Motif’s Epigenetic Services protocols have reduced starting material requirements by 1,000-fold to enable our customers access to data sets never before achievable.
LOW CELL ChIP-SEQgenerate genome-wide binding profiles from as few as 10,000 cells
USE FOR ANALYSIS OF:
• Magnetic or FACS sorted cells
• Primary progenitors, Beta cells, T cells, etc.
• Small clinical biopsies
TARGETS:
• Histone modifications - 10K cells
• Transcription Factors - 100K cells
• Chromatin Modifiers - 100K cells
IDEAL FOR LIMITED SAMPLE TYPES
For more information about Low Cell Number ChIP-Seq Services, please visit us at www.activemotif.com/lowcellcs.
FIGURE 1: A) Publicly available CTCF ChIP-Seq data set
from 10,000,000 cells. B) Active Motif’s Low Cell CTCF
ChIP-Seq data set from 50,000 cells and (C) 10,000 cells.
FIGURE 2: A) Publicly available H3K27me3 ChIP-Seq data set
from 10,000,000 cells. B) Active Motif’s Low Cell H3K27me3
ChIP-Seq data set from 50,000 cells and (C) 10,000 cells.
Fix samples, preparechromatin and sonicate
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Construct ChIP-Seq libraries
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Low Cell ChIP-Seq ServicesCustomers submit cell pellets or frozen tissues, then we:
Perform ChIP with a ChIP-validated antibody
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Perform Next-Generation Sequencing
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Perform data analysis5
Publicly available ChIP-Seq data sets generated using 10 million cells are comparable to data sets generated with Active Motif’s Low Cell ChIP-Seq pipeline using much lower amounts of starting material.
LOW CELL ChIP-SEQ PRODUCES COMPARABLE DATA TO STANDARD ChIP-SEQ
Low Cell Number ChIP-Seq is offered as an end-to-end service, covering every step from chromatin preparation to comprehensive bioinformatic analysis.
Just send us your frozen cells or tissue biopsy samples and we’ll do the rest.
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ChIP ANTIBODY VALIDATIONservices to test the suitability of your antibody for ChIP applications
One of the greatest challenges in ChIP experiments is the lack of available antibodies that can recognize fixed, target-bound proteins and that function in immunoprecipitation. Active Motif’s ChIP Antibody Validation Service makes ChIP antibody validation simple, fast and convenient. Let the ChIP Experts® do the work for you.
Our Epigenetic Services team specializes in performing ChIP-Seq and our in-house validation program has ChIP-qualified antibodies for hundreds of different targets. If your target of interest is on our list, we can start your project immediately. Otherwise, submit your antibody to us for ChIP validation and our Antibody Validation Service can give you an answer in as little as 3 weeks.
Customer submits fixed cells or frozen tissue
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ChIP-Seq library construction
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Next-Generation Sequencing
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Data analysis and delivery6
ChIP reactions3
Chromatin preparation2
ChIP ANTIBODY VALIDATION SERVICES
PEAKS Antibody passed its validation.
NO PEAKS Antibody failed its validation.
• Submit any antibody for testing
• ‘Yes’ or ‘No’ results for ChIP-Seq functionality
• Results in 3-4 weeks
• Hundreds of antibodies already validated
For more complete information, visit us at www.activemotif.com/ab-val.
FIGURE 1: BRD4 and H3K27ac ChIP-Seq data identify super-enhancers. The super-enhancer is defined
by the clustering of high intensity peaks (copper hashes). This super-enhancer is marked by high intensity
BRD4 and H3K27ac ChIP-Seq signal.
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FIGURE 2: Identification of super-enhancers. Enhancers are plotted
in increasing order based on ChIP-Seq peak intensity using the
ROSE algorithm. Super-enhancers are the population above the
inflection point of the curve.
SUPER-ENHANCER PROFILINGspecialized ChIP-Seq data generation and analysis services for genome-wide super-enhancer profiling
Active Motif offers specialized ChIP-Seq services for super-enhancer identification and analysis. The service provides a complete solution for genome-wide super-enhancer profiling, from sample preparation to data delivery. We utilize validated antibodies to enable the most comprehensive analysis of these master regulatory domains.
USE SUPER-ENHANCER SERVICES TO IDENTIFY:
• Master regulators of cell identity
• Regulatory regions associated with disease
• Mechanisms of BRD4 inhibitors
To learn more, visit www.activemotif.com/services-superenhancer.
Simplify genome-wide super-enhancer analysis by letting Active Motif do the work for you.
VALIDATED ANTIBODIES FOR SUPER-ENHANCER PROFILING
H3K27ac BRD4 H3K4me1 OCT4 SOX2 MED1 NANOG
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ChIP-SEQ SPIKE-INa novel ChIP-Seq normalization strategy to reveal latent and subtle biological effects
Active Motif’s Services team has recently created and validated a ChIP-Seq Spike-in Normalization strategy that can correct for variance. Spike-in is now available as part of our end-to-end ChIP-Seq Service.
HOW DOES IT WORK?ChIP-Seq reactions: •A standard ChIP-Seq reaction is set
up using your experimental chromatin and antibody of interest.
•Drosophila melanogaster chromatin is “spiked in” to each reaction as a minor fraction of total chromatin.
•An antibody recognizing the Drosophila-specific histone variant, H2Av, is added to the reaction to reliably pull down a small fraction of Drosophila chromatin.
•Following ChIP, immunoprecipitated DNA sequences are analyzed by Next-Generation Sequencing (NGS).
As a leader in ChIP innovation, Active Motif has developed ChIP-Seq Spike-in, an instrumental technical advancement in ChIP that enables more accurate sample comparisons and reveals latent biological effects. The normalization strategy is universally applicable to any ChIP experimental set-up.
Normalization: •Following NGS, sequence tags are
aligned to the experimental refer-ence genome (e.g. human) and the Drosophila genome.
•Variances in Drosophila tag counts are equalized across samples.
•The same ratio used to equalize Drosophila tag counts is applied to human tag counts for normalization.
ADVANCED ChIP-SEQ NORMALIZATION ChIP is a multi-step process in which the effects of sample loss, uneven sequencing read depth or technical variation can often lead to uninterpretable results or conceal subtle or latent biological effects in your samples.
Results: Biases introduced during Next- Generation library amplification and sequencing also occur in the Drosophila spike-in chromatin. Normalization using our spike-in strategy eliminates these biases to enable the observation of any significant biological changes in your ChIP-Seq samples (see ChIP-Seq Normalization Workflow, Figure 1, opposite page). To learn more, please visit us at www.activemotif.com/services-normalize.
• Uncover latent or subtle biological effects
• Monitor consistency between samples
• Reduce effects of technical variation
• Eliminate sample bias
CHIP-SEQ NORMALIZATION ADVANTAGE
ExperimentalChromatin
Spike-inChromatin
Antibody ofinterest
Spike-inAntibody
Chromatin Immunoprecipitation
Sequencing
Map to Drosophilagenome
Map to experimentalgenome
Normalize Drosophilatag counts across samples
Normalize sample tag counts by same ratio
A standard ChIP reaction is set up using experimental chromatin and an antibody of interest. Drosophila spike-in chromatin and a spike-in antibody that recognizes the Drosophila chromatin are also added to the reaction. Since variation introduced during the ChIP procedure will also occur with the immunoprecipitated spike-in chromatin, the spike-in signal can serve as a reference to normalize the test sample signals.
CHIP-SEQ NORMALIZATION WORKFLOW
Active Motif’s ChIP-Seq Spike-in Normalization strategy reveals EZH2 inhibitor-induced changes in H3K27me3 levels that were previously undetected using a standard ChIP-Seq protocol.
UNCOVER LATENT BIOLOGICAL EFFECTS OF COMPOUND TREATMENTS
FIGURE: Cells treated with a small molecule inhibitor of EZH2 methyltransferase
have dramatic reductions in global H3K27me3 levels. However, H3K27me3
ChIP-Seq using standard ChIP-Seq protocols (-) does not detect these differences.
Incorporation of Active Motif’s ChIP-Seq Spike-in Strategy (+) reveals the
expected decrease in H3K27me3 ChIP-Seq signal.
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GENE EXPRESSION SERVICESservices for whole transcriptome sequencing and gene expression analysis
Active Motif transcriptome analysis services include RNA-Seq for identification and quantitation of RNA transcripts and RNA Pol II ChIP-Seq for quantitation of transcription rates to enable rapid profiling of changes in gene expression associated with transcription factor (TF) and histone modification occupancy.
RNA-SEQ SERVICESSimply submit RNA, cell or tissue samples. Order RNA-Seq alone or combine with ChIP-Seq data to uncover contextual information about:
• Differential gene expression• Changes in gene structure or splicing patterns• Phenotypic effects of TF binding on gene expression
RNA-SEQ SERVICES FEATURES
• PolyA selection or rRNA reduction • QC performed using Bioanalyzer• Directional library preparation • Data analysis options (TOPHAT,
• 50bp or 100bp paired-end reads CUFFLINKS, CUFFCOMPARE, CUFFDIFF)
RNA POL II ChIP-SEQ SERVICESAnalysis of RNA Pol II occupancy as a proxy measurement of transcription rates offers the advantage of enabling you to:
• Measure transcription without the influence of RNA half-life• Identify genes poised for transcriptional activation• Generate gene expression data from cells used for ChIP-Seq• Measure changes at early time points post-treatment
Prepare RNA from cells or tissues
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Construct directionallibraries
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Generate cDNA2
Perform Next-Generation Sequencing
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Perform data analysis5
RNA-SEQ SERVICES
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Igf1r, Gene Length = 281,411 bp
FIGURE: Gene expression profiles vary depending on the analysis
method. Data for Igf1r was extracted from RNA–Seq and RNA Pol II
ChIP-Seq data sets. Cell treatment resulted in induced gene
expression that was measured at various time points. The
cumulative data show that, as measured by RNA Pol II ChIP-Seq,
transcription is induced immediately, while mRNA levels only
accumulate over time.To learn more, visit www.activemotif.com/services-expression.
FIGURE 1: Above, Next-generation sequencing data correlates with CpG density.
Right, Genome-wide profiling of DNA variants.
GENOME-WIDE DNA METHYLATION PROFILINGActive Motif provides comprehensive services for DNA enrichment and profiling of genomic regions marked by DNA methylation or DNA methylation variants. DNA is enriched using our highly validated collection of methylation or variant specific antibodies followed by sequencing on an Illumina next-generation sequencing platform for genome-wide profiling of these modifications.
FIGURE 2: Single base-pair validation of differentially methylated
sites identified by MeDIP. Heat map represents Targeted Next-
Generation Bisulfite Sequencing data of 72 CpGs from nine samples.
Differential methylation is observed across the samples.
DNA METHYLATION SERVICESservices for whole-genome and gene-targeted DNA methylation analysis
hmC
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MeDIP CpG Density
TARGETED BISULFITE SEQUENCINGOnce differentially methylated regions are identified as potential biomarker candidates or regions of interest, these regions require further validation across larger populations. Active Motif’s Targeted Next-Generation Bisulfite Sequencing Service offers a single base-pair, high-throughput solution for targeted validation of these regions. Services include:
| Bisulfite conversion | Primer design and testing | PCR amplification| Barcoded library generation| DNA sequencing| Data analysis
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For a complete list of services, visit www.activemotif.com/services-methylation.
DNA PROFILING AVAILABLE FOR:
| 5-Methylcytosine (5-mC)
| 5-Hydroxymethylcytosine (5-hmC)
| 5-Formylcytosine (5-fC)
| 5-Carboxylcytosine (5-caC)
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RIME differs from traditional IP approaches because the starting material has been cross-linked, offering the advantage of:
1) targeting only DNA, chromatin and associated proteins,
2) enabling capture of low affinity interactions which are lost in IP/mass spectrometry,
3) allowing more stringent wash conditions resulting in less non-specific interactions, and
4) stabilizing protein-DNA interactions to enable the capture of proteins that are bound at adjacent sites in the DNA and are independent of protein-protein interactions.
IDENTIFY INTERACTOMES REGULATING GENE EXPRESSIONGene regulation is often oversimplified when the focus is on one particular transcription factor (TF) in any given cell model. In reality, differential gene expression is greatly influenced by cofactors and other protein interactions with chromatin. RIME sorts out this complexity by providing a means to identify the protein interactions that are important for gene regulation. WHY RIME?Immunoprecipitation (IP) followed by mass spectrometry has traditionally been the approach used to identify proteins interacting with a target of interest.
RIME ADVANTAGE
• Identify transcriptional cofactors
• Identify TFs bound to DNA at sites adjacent to your target TF
• Identify important targets for ChIP-Seq analysis
• Detect low affinity interacting proteins
Nuclear isolation and sonication
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Purification of proteins and tryptic digestion
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Mass spectrometry4
Immunoprecipitation2
RIME SERVICES
The newest addition to Active Motif’s suite of epigenetic and gene regulation focused services, RIME (Rapid Immunoprecipitation Mass Spectrometry of Endogenous Proteins) sheds light on the complex process of gene regulation by enabling the capture and identification of interactomes, or the associated protein networks, of an endogenous protein of interest.
INTERACTOME PROFILINGmass spectrometry service for identification and characterization of protein interactomes
For more information about RIME Services, please visit us at www.activemotif.com/rime.
The RIME service includes immunoprecipitation of a target protein from cross-linked cell nuclei followed by mass spectrometry and data analysis.
RIME combines immunoprecipitation and mass spectrometry of a target protein for identification of endogenous interacting proteins in formaldehyde cross-linked cells.
500 µg chromatin starting material: 48% coverage
50 µg chromatin starting material: 43% coverage
Cross-link cells Isolate nuclei Sonicate
Immunoprecipitate target
Identify interactingproteins by mass spectrometry
RIME SERVICE WORKFLOW
RIME was performed to identify proteins that interact with the RNA polymerase transcriptional machinery. Immunoprecipitations were performed with an antibody against POLR2A and two different amounts of cross-linked chromatin from MCF7 cells. Immunoprecipitated proteins were measured by mass spectrometry.
CHARACTERIZATION OF ENDOGENOUS INTERACTOMES BY RIME
500 µg Chromatin 50 µg Chromatin
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*Peptides in an IgG negative control have been subtracted out
FIGURE 1: Venn Diagram of RIME data from MCF7 cells
shows strong overlap of the detected proteins even in
experiments using 10-fold greater amounts of chromatin
starting material.
FIGURE 2: One indicator of a successful RIME experiment
is detection of the target peptide at high levels.
Here, POLR2A peptide is detected from both low and
high amounts of chromatin starting material.
TOP POLR2A INTERACTING PROTEINS IDENTIFIED BY RIME• DNA-directed RNA polymerase II subunit RPB1 (POLR2A)•Transcription elongation factor SPT6 (SUPT6H)• DNA-directed RNA polymerase II subunit RPB2 (POLR2B)• Transcription elongation factor SPT5 (SUPT5H)• FACT complex subunit SPT16 (SUPT16H)• Regulation of nuclear pre-mRNA domain-containing protein 2 (RPRD2)• E3 ubiquitin-protein ligase TRIM33 (TRIM33)• RNA polymerase II-associated factor 1 homolog (PAF1)• RNA polymerase-associated protein CTR9 homolog (CTR9)• PHD finger protein 3 (PHF3) Poly [ADP-ribose] polymerase 1 (PARP1)• RNA-binding protein 25 (RBM25)• DNA replication licensing factor MCM7 (MCM7)• Putative pre-mRNA splicing factor ATP-dependent RNA helicase DHX15 (DHX15).
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To help our clients interpret large and highly complex whole-genome data sets, our Services team provides complete bioinformatic analysis and support for all services. We leverage our extensive experience and background to extract relevant information embedded within those complexes data sets and provide you with the most meaningful and highest quality data analysis.
BIOINFORMATIC SOLUTIONScomprehensive and customizable data analysis and support from our expert team of scientists
| Data filtering
| Genomic alignment
| Peak calling
| Tabulated peak information
| Genomic region pie chart
| Sample comparison scatter plots
| Average peak signal graphic
| Heat maps
| Average peak intensity box plots
| Sample comparison Venn diagrams
| Motif enrichment analysis
| Browser tracks for visualization
STANDARD ANALYSIS
For more information, please visit us at www.activemotif.com/services.
• Standard analysis included in every assay
• Custom analysis also available
• Support from expert scientists
• Delivery of the highest quality data
The LightSwitch™ Reporter Assay System provides a comprehensive genome-wide approach for studying gene element function and identifying gene regulatory networks. In addition to over 30,000 ready-to-use promoter & 3´UTR reporter constructs, Active Motif offers services for custom cloning and mutagenesis, miRNA target validation, sequence variant analysis and pathway screening.
Active Motif’s LightSwitch™ Reporter Assay System is a complete solution for performing gene regulation studies. All LightSwitch vectors utilize our RenSP luciferase engineered to produce higher levels of luminescence with shorter half-life than other Renilla luciferases. LightSwitch reporter vectors provide industry-leading sensitivity and dynamic range, guaranteeing the best results possible. To learn more, please visit us at www.activemotif.com/lightswitch.
TRANSCRIPTIONAL REGULATION
• Assess promoter and enhancer response to transcription factor modulation
• Confirm functionality of binding sites identified in ChIP-Seq experiments
• Measure functional effects of mutated motifs
• Validate computational predictions and add biological relevance to microarray or NGS data
LIGHTSWITCH™ SYSTEM APPLICATIONS
POST-TRANSCRIPTIONAL / miRNA REGULATION
• Validate the 3´UTR targets of any miRNA
• Measure the effect of miRNA or siRNA on transcript stability or translation efficiency
• Quantify the impact of a 3´UTR in post-transcriptional gene regulation
• Assess miRNA function in response to various stimuli
GENE REGULATION SERVICESLightSwitch™ comprehensive suite of services to accelerate your functional genomics research
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Active Motif’s Custom Cloning & Mutagenesis Services offer a complete high-throughput solution for gene regulation studies in living mammalian cells and functional validation of protein binding events detected in ChIP assays. Simply submit the sequence information for your genomic elements of interest, and in 4-6 weeks, we will deliver transfection-ready luciferase reporter vectors for use in your assay experiments.
CUSTOM CLONING SERVICEIn addition to the 30,000 human regulatory elements available as pre-cloned LightSwitch reporter vectors, Active Motif offers custom services to clone and sequence validate any human, mouse or rat genomic element into any LightSwitch reporter vector. LightSwitch vectors are engineered to provide optimal kinetics from your reporter assays, Figure 1, opposite page.
CUSTOM MUTAGENESIS SERVICEActive Motif also offers a comprehensive mutagenesis service to create sequence variants of any genomic sequence to enable you to map functional motifs or characterize the effects of sequence changes on gene expression.
CUSTOM CLONING & MUTAGENESISservices to clone and sequence validate your elements of interest in any LightSwitch™ luciferase vector
SERVICE FEATURES
• Clone any genomic element of interest
• Promoter, long-range, 3’UTR and 5’UTR reporter vectors available
• Ideal for gene regulation studies or validation of ChIP binding events
• Create sequence variants of any genomic sequence
• Custom vectors ready in 4-6 weeks
For more on Custom Cloning & Mutagenesis Services or to place an order, please visit www.activemotif.com/ls-services.
INSERT ANY ELEMENT... FROM... INTO ANY LIGHTSWITCH VECTOR
Promoters Rat Promoter
Enhancers Mouse Long-range
UTRs Human 3’UTR
Motifs 5’UTR
SNP variants
FIGURE 1: Map of empty LightSwitch Promoter and
3´UTR reporter vectors showing the multiple cloning
site (MCS) for insertion of genomic elements. In addition,
long-range, and 5´UTR reporter vectors are available. All
LightSwitch luciferase vectors utilize RenSP, an optimized
Renilla luciferase gene that has been engineered to
yield quantitative measurements with industry-leading
sensitivity and dynamic range.
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ANY GENOMIC ELEMENT... INTO ANY LIGHTSWITCH VECTOR
The backbone of the LightSwitch System is the LightSwitch reporter vectors that utilize our RenSP luciferase technology to provide optimal brightness and sensitivity for superior quantitation. Services include cloning and sequence validation of every reporter construct.
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FIGURE 2: LightSwitch 3´UTR Reporter
constructs containing the 3´UTRs of 6
genes known to be miR-122 target sites
were subjected to site-directed mutagenesis.
Constructs were co-transfected in
cells with an miR-122 miRNA mimic or
non-targeting control and then assayed
to identify seed sequences necessary for
miR-122 function. Wild-type miRNAs reduce
luciferase expression through 3´UTR
interactions. miRNA mutations in the
seed sequence disrupt miRNA targeting
and reduce the functional effect.
Active Motif’s LightSwitch™ reporter vectors are engineered to provide industry-leading sensitivity and dynamic range for optimal results from your reporter assays.
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Active Motif’s Custom Cloning & Mutagenesis Services not only enable you to assess the activity of regulatory elements, but also map functional motifs through site-directed mutagenesis and creation of sequence variants for identification of seed sequences necessary for function.
IDENTIFY SEQUENCE MOTIFS NECESSARY FOR FUNCTION
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Identifying the mRNA targets of an miRNA in any given cell type remains a significant challenge. Computational predictions are not accurate and require validation while transcript-based expression analysis cannot measure the functional role of miRNAs. Our luciferase-based 3´UTR reporter screening service has the ability to measure actual miRNA function.
Our miRNA Target Validation Service also includes a series of complementary high-throughput approaches that enable you to assess:
• Dose-dependency• Specificity• Reproducibility
To learn more about miRNA Target Validation Services, please visit us at www.activemotif.com/ls-services.
SERVICE APPLICATIONS
• Identify functional miRNA targets
• Compare luciferase data with endogenous transcript and protein levels
• Characterize dose responsiveness of luciferase reporters
• Verify specificity of miRNA/reporter interaction
3’UTRReporter
Hybrid LUC-3UTRTranscript
promoterluciferase 3’UTR
miRNA mimicor
non-targeting control
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1- 3́ UTR constructs are transfected into your cell line
2- A constitutive promoter drives production of a hybrid RenSP-3́ UTR transcript
3- Optimized LightSwitch reagents are used to achieve maximum sensitivity and dynamic range
4- The effect of the 3´UTR on the hybrid transcripts stability and/or translation efficiency is measured
5- Co-transfected synthetic miRNA or miRNA inhibitor included to measure its impact on miRNA function
6- Once targets are identified, Sequence Variant Assay Services (page 18) can be used to assess the impact of sequence variation on 3́ UTR activity
miRNA TARGET VALIDATION SERVICE
miRNA TARGET VALIDATIONfull screening service to validate the 3´UTR targets of miRNAs of interest
FIGURE: Results from functional screen of predicted miR-122 targets in HT-1080 cells.
A) The log2 ratio observed for each tested 3´UTR reporter in the presence of the miR-122 mimic
over the luminescence when transfected with the non-targeting control shows 40.8% of the
predicted targets were significantly altered in the mimic co-transfection. Furthermore, of those
3´UTRs with significantly altered luminescence, 43.1% were repressed 2-fold or more by the
miR-122 mimic.
B) Highly repressed targets from the above screen were tested at miRNA concentrations ranging from
0.0625 to 100 nM. The tested 3´UTR reporters responded to miR-122 mimic in a dose-dependent
fashion, with EC50 at or below 1.5 nM.
C) Interaction of miR-122 with 3´UTR targets was evaluated. 3´UTR targets of miR-122 were
cloned into the LightSwitch UTR vector and co-transfected with either miR-122 mimic or a
non-targeting control miRNA in K-562 cells. The graph shows the four strongest responders
along with two non-responding controls.
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IDENTIFY FUNCTIONAL TARGETS OF YOUR miRNA OF INTEREST
Active Motif’s miRNA target screening service leverages our 3´UTR clone collection to screen hundreds of putative miRNA targets. Utilize our expertise in cloning and reporter assays to accelerate your miRNA studies.
Active Motif’s miRNA Target Validation Service uses our genome-wide collection of cloned human 3́ UTRs to conduct high-throughput, cell-based assays to validate the targets of your miRNAs of interest.
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Once you have identified a regulatory element of interest, our Custom Services team can perform site-directed mutagenesis or custom cloning of haplotypes, then conduct reporter assays to quantify the functional impact of sequence variation on promoter or 3´UTR activity.
Simply specify the sequence variants you want to have created. Our service team will create variant versions of the promoters or 3´UTRs in a LightSwitch reporter vector and conduct transient reporter assays to quantify the effects of the sequence changes and assess the functional impact of sequence variation on promoter or 3´UTR activity.
For more information on Sequence Variant Assay Services, please visit us at www.activemotif.com/ls-services.
SEQUENCE VARIANT ASSAY SERVICE WORKFLOW
YOU:
• Identify the promoters and 3´UTRs of interest
• Specify the variants you want to have created
WE:
• Create variant versions of the promoters or 3´UTRs in a LightSwitch reporter vector
• Transfect the reporter constructs into living cells
• Measure luciferase activity and process the data
SERVICE APPLICATIONS
• Study the effects of naturally occurring sequence variation
• Create specific mutations in motifs, transcription factor binding sites or putative miRNA targets
• Analyze individual SNPs or mutated motifs
• Create clones from individual genomic samples for haplotype studies
LightSignal
PromoterCloned into Reporter Vector
RenSPLuciferase
RenSPLuciferase
LightSwitch™PromoterReporter
Gene of interest 3´UTR
LightSwitch™3´UTR
Reporter
Cloned into Reporter Vector
Hybrid LUC-3´UTRTranscript Light
Signal
SERVICES WORKFLOW
SEQUENCE VARIANT ASSAY SERVICESassess the functional impact of sequence variation on promoter or 3´UTR activity
Active Motif provides a cell-based phenotypic screening service to measure the effects of your test compounds on a variety of biological pathways. We offer readouts for 48 validated human promoters to measure gene expression changes associated with 11 disease-related pathways. The service enables you to screen full regulatory networks and create pathway activity profiles.
• Analyze 11 pathways in an HTS environment
• Perform lead compound validation/optimization, mechanism of action, and off-target analysis
• Evaluate dose-response and analyze trends
• Secondary screening, “Hits-to-Lead”
LightSwitch Pathway Screening Services can profile pathway responses to treatments. The heat map on the right shows the inducible activity of 29 different reporter constructs that represent 10 different pathways in response to 15 different treatments.
QUANTITATIVE PROFILING OF COMPOUND EFFECTS ON RELEVANT PATHWAYS
To learn more about LightSwitch Custom Services, please visit us at www.activemotif.com/ls-services.
DISEASE PROFILING PANEL48 promoters and controls
Pathway activity readout for:
HIF-1a Hypoxia
NFkB Inflammation
CREB cyclic AMP
HSF1 Heat shock
p53 DNA damage, apoptosis
STAT Interferon
SREBP Cholesterol biosynthesis
ER Estrogen
AR Androgen
GR Glucocorticoid
AhR Toxicity
TCD
D
DM
SO
1% O
2
PM
A
Hea
t sho
ck
IFNa
Lova
stat
in
R18
81
Pre
dnis
one
TNF-
a
Nut
lin
Estr
adio
l
DFO
IFNg
Synt
heco
lCell Treatments
Representative R
eporter Constructs
Tox/Ahr
Controls
log2 ratio treated/untreated
Androgen
CREB
Estrogen
p53
Hypoxia
STAT
Heat shock
NFkB
Glucocorticoid
PATHWAY SCREENING SERVICESusing our collection of validated assays to measure pathway activation associated with a number of diseases
20 www.activemotif.com
HISTONE PTM MULTIPLEX SERVICE
quantify histone modifications in multiplex using Luminex® xMAP® technology
Active Motif now offers Custom Services for our Histone H3 PTM Multiplex Assay. The service uses Luminex® xMAP® magnetic bead-based multiplexing technology to simultaneously measure multiple histone modification targets in a single reaction. This first-of-its-kind multiplex epigenetic assay generates more data with less sample than traditional Western blot or ELISA methods.
THE SUPERIOR ASSAY CHOICE TO SCREEN EPIGENETIC VARIATIONProfiling variances in clinical or compound-treated samples using traditional Western blots or ELISAS requires large amounts of samples that are already in scarce supply. Traditional assays are also limited by low through-put and the inability to simultaneously analyze multiple targets. Our Histone H3 PTM Multiplex Service provides a rapid, highly sensitive method to enable profiling of changes in histone modification levels in multiplex. The assay’s ability to multiplex analysis of specific and off-target effects and its minimal cell number requirement makes it ideally suited for screening and profiling of clinical or compound-treated samples.
The Histone H3 PTM Multiplex Service gives you access to ground-breaking technology that enables high-throughput multiplexed screening of histone modifications. The end-to-end, customizable service includes sample preparation, assay design and execution, data analysis and support. Our expert scientists will consult with you on project-specific details and outline an appropriate experimental design.
ASSAY FEATURES
• MULTIPLEX – perform multiplexed histone modification analysis
• EFFICIENT – use less input amounts than WB or ELISAs
• SENSITIVE – can detect signal from as little as 500 ng per well
• HIGH CONTENT – simultaneously measure specific & off-target effects
AVAILABLE TARGETS
H3 Total H3 pan-ac
H3K4me3 H3S10ph
H3K9ac H3T11ph
H3K9me1 H3K27ac
H3K9me2 H3K27me2
H3K9me3 H3K27me3
H3K56ac H3K36me3
For more information, please visit us at www.activemotif.com/services-luminex.
Histone-specific modifications in the N-terminus are captured using antibodies conjugated to fluorescent-labeled magnetic beads. Each bead emits a unique fluorescent signal to enable detection of multiple targets within the same well. A biotinylated H3 C-terminal antibody is used to capture histones. Streptavidin-phycoerythrin is then added to bind biotin and produce a signal. The Luminex instrument is used to read signals and decipher both the bead identity and the number of binding events pertaining to each histone modification.
HISTONE PTM MULTIPLEX ASSAY WORKFLOW
FIGURE: The Histone H3 PTM Multiplex Assay shows increased histone acetylation in response to SAHA-mediated HDAC inhibition. HeLa cells treated with increasing concentrations of the HDAC
inhibitor SAHA were evaluated in a multiplex of H3 pan-acetyl, H3S10ph, H3K9ac, H3K4me3, H3K27me2 & H3 Total Ab-conjugated beads using the Histone H3 PTM Multiplex Assay. Total H3 beads
were used for normalization. The results demonstrate the ability of the assay to simultaneously assess specific and off-target effects of the treatment on histone modification levels. The dashed lines
represent IC50 values, 4.0 µM and 4.6 µM, determined for pan-acetyl and H3K9ac, respectively.
Streptavidin-phycoerythrin
Biotinylated Histone H3 Ab
Histone containing PTM
PTM Ab-conjugated bead
Ac
H3
SA-PE
Me3
H3
SA-PE
Biotin
P
BiotinBiotin
SA-PE
H3
The Histone H3 PTM Service’s multiplexing and sample throughput capabilities are ideally suited for screening and profiling epigenetic variances caused by disease or compound treatment. Multiplexing has the added advantage of enabling simultaneous analysis of both specific and off-target effects.
PROFILE CHANGES IN HISTONE MODIFICATIONS IN CLINICAL AND COMPOUND-TREATED SAMPLES
2K
4K
8K
6K
10K
-2 -1 0 1 2log[SAHA, µM]
H3K4me3
H3S10ph
H3K27me3
Med
ian
Fluo
resc
ent
Inte
nsity
(MFI
)
6K
8K
10K
12K
-2 -1 0 1 2log[SAHA, µM]
H3K9ac
H3 pan-acetyl
Med
ian
Fluo
resc
ent
Inte
nsity
(MFI
)
SPECIFIC EFFECTS OFF-TARGET EFFECTS
Enabling Epigenetics Research
NORTH AMERICA
Toll Free: 877 222 9543Direct: 760 431 1263Fax: 760 431 [email protected][email protected]
Customer Service: [email protected]
JAPAN
Direct: +81 (0)3 5225 3638Fax: +81 (0)3 5261 [email protected]
EUROPE
GERMANY 0800/181 99 10UNITED KINGDOM 0800/169 31 47FRANCE 0800/90 99 79OTHER COUNTRIES, DIRECT +32 (0)2 653 0001Fax: +32 (0)2 653 [email protected]
CHINA
Hotline: 400 018 8123 Direct: +86 21 2092 [email protected]
