![]() “This is the perfect high-coverage concealer, yet looks natural and luminous," says Tom Pecheux, Global Beauty Director for YSL Beauté, in a release.Īvailable in 16 shades, the Touche Éclat High Cover includes soft focus fillers and light-generating pigments, while also offering a high concentration of makeup pigments for long-wear coverage. Here are our dupes for YSL Touche Eclat - Radiant Touch, which is a concealer with a luminous finish and sheer coverage that retails for 40.00 and contains 0.1 oz. Face Primer Yves Saint Laurent Touche Eclat Radiant Touch Yves Saint Laurent 3.7 908 reviews 64 would repurchase 4.1/5 package quality price range ingredients AQUA (WATER). Yes, the Touche Éclat High Cover Radiance Concealer is ideal for anyone who loves the illuminating power of the original but has always wished it offered more coverage. Cosmetics’ new 5 Flawless Brightening Concealer was a spot-on dupe for YSL’s 38 Touche Éclat. Touche Éclat All Hours Primer Foundation Makeup Concealer Highlighter Blush & Bronzer Powder NU Makeup Sets Refine PRICE RANGE £20.00 £200. So, we're sure fans of the brand will be as thrilled as we were to hear that YSL is now launching a brand new version of Touche Éclat for 2019 – and, this time, it really is a concealer, designed to add radiance while also offering an extra dose of colour to your complexion. On the thread, multiple concealer-passionate users swore that e.l.f. While many of us think of that iconic golden pen as an under-eye concealer, you might be surprised to hear that the Touche Éclat is actually a highlighter, designed to illuminate the skin and enhance your features. Check out similar products based on the Style of Touche clat High Cover Radiant Under-Eye Concealer that is touch of. ![]() Use shade 01 porecelain Answered by: LESLEY. Yves Saint Laurent Touche Eclat Radiant Touch Concealer Luminous Sand for Women, 0.1 Ounce. ![]() The original YSL Beauté Touche Éclat Radiant Touch has been a cult beauty product pretty much since it launched back in 1992. Whish shade should I use for the YSL touche eclat high cover concealer and the YSL touche eclat highlighter please Asked by: Manon. ![]()
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![]() That's why it's one of the best streaming services. So, that makes it really easy to recommend where cord-cutters should turn to watch the NFL season: Fubo TV. And Fubo is the best place to watch the Super Bowl in 4K.īuilt for all things sports, Fubo has all three of those local broadcasters, plus ESPN and NFL Network. During the regular season, local and primetime games will be included in NFL Plus. But if watching solely on your mobile device is OK, then this $4.99 per month service allows you to watch live games on your phone or tablet. ![]() The newest competitor for streams this season is NFL Plus, but it's just for mobile devices and tablets. Amazon Prime Video has some Thursday Night Football games, while the NFL Network will continue to offer a bunch of games throughout the year. CBS gets AFC games on Sunday afternoons, and FOX will have the NFC games. Just like last season, ESPN has Monday Night Football and NBC will have Sunday Night Football. Looking at the NFL 2022-23 schedule, you can see that games will air on a wide range of services and channels, including old standbys like ESPN, NFL Network and your local FOX, NBC and CBS affiliates. We've also got all the intel on how to watch USWNT live streams. Here are the best ways to live stream NFL games. With so many good games, you're probably looking for a way to catch the action online. The methods for watching NFL games change often as the league signs new contracts and streaming services change their policies, so we're always updating this guide with the latest broadcast info, as well as the current week's schedule. And if you only want local games on your phone, well, NFL Plus may be of interest. We've got everything you need to stream this NFL season from a phone, laptop, set-top box or anything in-between. Thanks to multiple streaming options, you have a wide array of ways to watch these NFL games. ![]() It was seemingly overly-filled with referee involvement. The Eagles had the easier-looking path in the Championship round, as the Chiefs three-point victory over the Bengals was tight. Watch anywhere: Try ExpressVPN risk-free.US channels: CBS/Paramount Plus, ESPN, Fox, NBC, NFL Network, NFL Plus and Prime Video. ![]() ![]() Optimizer row goal information in query execution plan added in SQL Server 2014, 20įIX: "sp_special_columns" returns incorrect result in SQL Server 20įIX: Slow restore performance when restoring a compressed backup on a disk with 4K sector size in SQL Server Update adds support for MAXDOP option for CREATE STATISTICS and UPDATE STATISTICS statements in SQL Server 2014, 20įIX: Automatic update of incremental statistics is delayed in SQL Server 2014, 20įIX: Deadlock occurs on single-user mode database when many connections access it simultaneously in SQL Server 20įIX: The tempdb system database is still encrypted after you turn off encryption on an instance of SQL ServerįIX: Execution stats of scalar user-defined function is added to Showplan XML file in SQL Server 2016 SP2 Update adds CPU timeout setting to Resource Governor workgroup REQUEST_MAX_CPU_TIME_SEC in SQL Server 2014, 20 Update adds a new column to DMV sys.dm_sql_referenced_entities in SQL Server 20 Update adds the "CLR strict security" feature to SQL ServerįIX: EXCEPTION_ACCESS_VIOLATION for query using sys.dm_os_memory_objects statement in SQL ServerįIX: Change Data Capture stops working after a recent cumulative update for SQL Server is installed Unified Showplan Schema for different versions of SQL Server Update adds overall query execution statistics information to the STATISTICS XML output in SQL Server 2012, 20įIX: Decreased performance and long waits for CLR_AUTO_EVENT and CMEMTHREAD when SQLCLR UDT is used as a stored procedure parameter for a SQL RPC callįIX: Data types of Query_hash and Query_hash_plan columns in 圎vents do not match the data types in DMVs sys.dm_exec_requests and sys.dm_exec_query_stats Update extends the Trace extended event with security protocol handshake information in SQL Server Information about enabled trace flags is added to the showplan XML in SQL ServerįIX: Memory grant that's required to run optimized nested loop join isn't reflected in Showplan XML in SQL ServerĪdds a stored procedure for the manual cleanup of the change tracking side table in SQL Server Informational messages added for tempdb configuration in the SQL Server error log in SQL Server 20 Update to add info about SQL Server startup account to security policy in SQL Server 20 error log ![]() Improved memory grant diagnostics using Extended Events in SQL Server 20įIX: Access violation occurs when a query references an SQLCLR function through a synonym in SQL Server 20 Improvement: Replace HTTP URLs with HTTPS URLs in SQL Server 2014 Installation Wizard to improve securityįIX: "Non-yielding Scheduler" error and SQL Server appears unresponsive in SQL Server 2014, 20 This list will be updated when more articles are released.įor more information about the bugs that are fixed and enhancements that are included in SQL Server 2014 SP3, see the following Microsoft Knowledge Base articles.įIX: New TCP connection is established and remains open until the process is terminated in SSIS 2014 In addition to the fixes that are listed in this article, SQL Server 2014 SP3 includes hotfixes that were included in SQL Server 2014 Cumulative Update 1 (CU1) through SQL Server 2014 SP2 CU13.Īdditional fixes that aren't documented here may also be included in the service pack. SQL Server 2014 SP3 upgrades all editions and service levels of SQL Server 2014 to SQL Server 2014 SP3. Microsoft SQL Server 2014 service packs are cumulative updates. List of improvements and fixes included in SQL Server 2014 SP3 ![]() SQL Server 2014 SP3 upgrades all editions and service levels of SQL Server 2014 through SQL Server 2014 SP3.įor more information about the cumulative updates that are available in SQL Server 2014, see SQL Server 2014 build versions. Note After you install the service pack, the SQL Server service version should be. ![]() SQL Server 2014 SP3 and Microsoft SQL Server 2014 SP3 Express are available for manual download and installation at the following Microsoft Download Center websites. More Information How to get SQL Server 2014 SP3 It includes all the information that you previously found in the release notes and Readme.txt files. ![]() Note This article serves as a single source of information to locate all documentation that's related to this service pack. It describes how to get the service pack, the list of fixes that are included in the service pack, known issues, and a list of copyright attributions for the product. This article contains important information to read before you install Microsoft SQL Server 2014 Service Pack 3 (SP3). ![]() ![]() ![]() The courier was a cheerful & professional fellow, who obviously enjoys his work. Although I didn’t track the parcel (I think it actually arrived before the tracking was activated), this was a good feature. The delivery time to our sleepy little hamlet of Bloemfontein was also remarkably quick- two days if I remember. If I might add just one small observation, the Giancarlo lamp colour in the checkout was white in the picture but labelled as black. The checkout & payment section was also easy to follow and intuitive. The website and catalogue is well laid out, logical & attractive, and I had no problems finding the lamps I wanted. This will be a very brief review, but it is all positive. ![]() The outdoor plug I bought was also broken and I had to exchange this at the Ruimsig store as well.ĭisappointing that the level of knowledge in lights are so poor at a place that specializes in it… I then went to Chamberlain’s hardware and they told me that I should’ve bought another part together with the dimmer plate for it to work – They then sold me a dimmer plate that worked for the LED lightbulbs. I exchanged the item at your Ruimsig branch and the salesperson again assured me that those dimmer plates will work for LED lightbulbs, but after I installed at home it didn’t work. After I received the order, I had to exchange the item because the two switches were for two different lights. The consultant also told me that the two switches on the dimmer plate is for the same light – One switch will turn the light on and off and the other switch will dim the light. I went onto your website and chatted to one of your consultants, who assured me that the dimmer plate I bought will work with LED lightbulbs. Come into my world, don’t force me to learn yours. ![]() I will know what I am looking for but I don’t speak your language. Do consumers know the different labels for your types of outdoor lights, for instance? I am intelligent and educated, but I don’t work in your industry so show me example photos of the various types of outdoor lights with your labels underneath. Think about using landing pages with examples of the lights rather than menu options. Don’t regard your web site as a catalogue, help your consumers through the process – try and replicate your in-store experience with your excellent sales staff. Personally I prefer the warmer end of the spectrum – but I had to google what that was in terms of K because you didn’t help me with that. You could also help us laymen with more information about light colour. You should cross link on your web site from light to bulbs, or bulb range appropriate to the light. But if I hadn’t, you wouldn’t have seen that sale as I would have gone to the most convenient solution for me – Builders up the road. Luckily I had some screw-in bulbs that were perfect for them. I recently ordered 3 desk lamps from you which arrived promptly without bulbs. Your light descriptions on the site are very sparse and don’t even tell me if the bulb is screw-in or bayonet. The one thing your sales team in-store are really good at – ensuring I walk out the store with bulbs that fit and create the lighting I want. Secondly, I think you are missing a big cross selling opportunity. There is a lot of flickering and instability with menus. Firstly, I normally do online shopping on my iPad and the experience around that could be improved a lot. I think you are missing a trick or two though. Your web site seems comprehensive in that there are many choices. Your staff were friendly, fast & helpful. Last update was on the 10th, so not sure if it is indeed out for delivery today. We completely understand a mistake creeping in, I am just frustrated by the lack of communication & the tracking site that does not seem to provide regular updates. The agent did indeed dispatch our lamp on 10 May, so we are awaiting confirmation from the courier as there has been no further updates on the tracking site. The wrong light would be collected and the correct one delivered, hopefully today. Again I followed up and your online agent swiftly replied and assured me that he would remedy the situation. It arrived on Tuesday 10 May, but once we opened the box, saw that our light bulbs were enclosed, but that a wrong light fitting was dispatched. I was then given confirmation that the order would arrive on Tuesday 10 May. I made contact on Monday 9 May in the morning, via the online chat, and was asked to send my email address and that there would be follow-up made.Įnd of day on Monday, I had still not received feedback so again I contacted you via the online chat. ![]() I checked the tracking number provided but after the 4th of May, there we no new updates. I placed an order on Monday 3 May for light bulbs & one standing lamp. ![]() ![]() ![]() A low figure (<10) indicates effective glare reduction. VLT is impacted by the openness factor, fabric color, and the amount of light that transmits through the fabric. Visible light transmittance (VLT): The percentage of the available visible light allowed to pass through the solar screen shade and window combined. Fabric color itself does not affect openness across a fabric product line. ![]() The more open the fabric, the more solar radiation passes through open space rather than fabric, affecting the overall performance and amount of glare protection, heat protection, and view-through. Openness factor (or Density): Measurement of the relative area of the fabric’s openings, or holes. The total value of all 3 percentages equals 100% of the available solar radiation. A low percentage indicates fabrics that absorb little solar energy.Īll of the available solar radiation is either transmitted through, absorbed by or reflected by the fabric. Solar absorptance (As): The proportion of solar energy absorbed by the fabric alone. A high percentage indicates fabrics that reflect solar energy well. Solar reflectance (Rs): The proportion of solar energy reflected away by the fabric alone. A low percentage indicates fabrics that reduce solar energy well. Solar transmittance (Ts): The proportion of solar energy transmitted through the fabric alone. The visible and invisible combined, or “spectrum”, make up what we see as light, feel as heat, and UV (ultraviolet) rays that are not seen or felt, but have damaging effects on skin, eyes and furnishings. Some of it is visible, some is not visible. What we call “sunlight” is the energy, or radiation that comes from the sun. Part of the information collectively known as “fenestration data” Understanding solar/optical/thermal factors ![]() ![]() There, you can connect to the Naperville Riverwalk ( ROUTE MAP). South of Blackwell, one can run on the W Branch DuPage River Trail for 5.4 miles ( ROUTE MAP) through Warrenville Grove Forest Preserve & the McDowell Grove Forest Preserve (7 miles of trails arund mature woodlands & open fields, TRAIL MAP), following the DuPage River as far as W Jefferson Ave. James Farm Forest Preserve and the larger Blackwell Forest Preserve, which features 7 miles of trails over rolling, hilly terrain around oak and hickory woodlands, prairies, grasslands, and wetlands ( Blackwell Trail Map). East of Danada, use the Prairie path alongside Butterfield Rd. Just east of Danada, the Morton Arboretum is fantastic for running, with its 9 miles of roads & 16 miles of trails. Park at Warrenville High School of Herrick Lake, and enjoy running in Herrick Lake Forest Preserve and the adjacent Danada Forest Preserve (a 2.8 mile limestone path connects them. A branch of the Illinois Prairie Trail connects several near-adjacent Forest Preserves, each of which have their own trails for running. This bike path is not as popular as some of the other, busier trails, so it’s a good choice for those seeking solitude. Paul Douglas Preserve: This massive Forest Preserve in Hoffman Estates is all open grasslands, wetlands, and resident birds, with a 7.5 mile paved trail looping through it. The 8.9 mile paved loop through the eastern end of the preserve is popular, or combine the unpaved yellow and brown trails for a 6.3 mile loop. Poplar Creek: In Elgin, west and just north of Chicago, the Poplar Creek Forest Preserve has various options for running through nice scenery and modest hills. For long runs, tack on more mileage on the Salt Creek Trail to the southeast. While there are plenty of running options on the 10.8 miles of trail here, the go-to choice is the 8 mile paved loop around the lakeshore. Schiller Woods INFOīusse Woods: The Ned Brown Preserve, better known as Busse Woods, is conveniently located at the intersection of I-90 and I-290 in Elk Grove. Make your own route or participate in one of Universal Sole’s Fun Runs or trail races. It mostly consists of picnic areas and playing fields, but runners will find access to myriad paths along the river here, with the option to extend or loop your route onto the Des Plaines River Trail. Schiller Woods: This bustling preserve to the northwest of downtown is actually within the Chicago city limits, and is incredibly popular for its proximity to O’Hare International Airport. The main loop around the park is 9.5 miles. It also boasts 11 miles of crushed limestone and grass trails. This hilly 2,503 acre property is known for its great views from the Rocky Glen Waterfall to the Sawmill Creek Bluff overlook. Waterfall Glen: Located in Darien, just southwest of Chicago, Waterfall Glen is the crown jewel of Illinois Forest Preserves. The most popular running route is the 5.2 mile paved circuit around Deer Grove East, although trail runners may enjoy a 5.4 mile unpaved loop around the west side of the park. The park is divided into the primarily wooded Deer Grove West, and the wetlands and prairies of Deer Grove East, divided by Quentin Road. North Branch Trail MAPĭeer Grove: The first of the Forest Preserves, is located in Palatine, about 40 miles northwest of the city. Runners can enjoy this preserve’s numerous wildflowers on a 1-mile section of the North Branch Trail (which extends north and south outside the preserve) or take on the unpaved trails east of the river ( Note: these can be quite muddy in the springtime). Harms Woods: North of Chicago in Glenview, Harms Woods is a 250-acre park divided by the Chicago River, and a favorite spot for outdoor exercise. A loop around the perimeter is 4.4 miles, with the option to extend your route south on the North Branch Trail. 5.9 miles of those paths are asphalt, so even pavement pounders can escape into nature. Skokie Lagoons: Paddlers and fishers flock to Skokie Lagoons to explore the vast network of pools and channels north of Chicago in Winnetka, but running is also popular on the 7.5 miles of trails here. ![]() See our overview map for a helpful guide by area. There are too many preserves to list them all here, but these are some of our favorites for running. ![]() Many have lengthy loops on paved or crushed stone paths, with opportunities to extend your run on nearby multi use paths. These beautiful green spaces throughout DuPage, Cook, Kane, Will, and Lake counties are popular for running. Get your fix of nature right in the Chicago suburbs with these Forest Preserve properties. ![]() ![]() ![]() ![]() You can use volume automation to work around this issue, but it’s a very valuable feature that most other DAWs do have. ![]()
![]() Dozens of original characters and story arcs will take you on a captivating journey. Swim, climb, vault, jump off or hoist up ledges as you traverse seamlessly through the world with a navigation system based on platformer expertise that breaks free from the classic bound-to-the-grid tileset movement. Along with timed hits, combat features multi-character combo attacks, boosting, and a strategic "locks" system offering options to play with different damage types in order to hinder enemies while they are channeling powerful attacks or spells, all in an immersive take on the genre no random encounters, no transitions to separate battlefields, and no grinding. All of our games taking place in a shared universe, we cannot wait to show you all the stories we can tell with the richness of the world and the sheer amount of characters that an RPG entails. To make a turn-based RPG that would pay homage to the greats of the 90’s, while bringing enough novelty to be its own thing. Promising the Sabotage touch in every system, Sea of Stars aims to modernize the classic RPG in turn-based combat, storytelling, exploration and interactions with the environment, while still offering a hearty slice of nostalgia and good old, simple fun.Ĭontinuing on the Sabotage journey of developing games that blend retro aesthetics and modern game design, invigorated and motivated by The Messenger’s overwhelmingly positive response, it was time for the real, big challenge. A prequel story set in The Messenger’s universe, it tells the story of two Children of the Solstice who will combine the powers of the sun and moon to perform Eclipse Magic, the only force capable of fighting off the monstrous creations of the evil alchemist known as The Fleshmancer. of Stars is a turn-based RPG inspired by the 90’s classics. To Garl, the call of adventure is an excuse to taste all the flavors life has to offer, and to challenge himself in preparing wholesome meals by making do with the daily unpredictabilities of being the devoted friend and caretaker of two Solstice Warriors on a mission. It was a leap of faith for some of you, but we promise to dedicate all of our energy into honoring all that support over the next 2 years. We want to thank everybody who supported the project so early. ![]() Sabotage Studio has also introduced us to Garl, who is a new playable character that will be joining the party in Sea of Stars, and will do anything to protect the Solstice Warriors Valere and Zale with three more characters still left to be revealed. Sea of Stars was actually successfully funded within hours after the Kickstarter campaign was announced, and over the past 29 days the developer has revealed that a bunch of stretch goals have also been reached - so there is plenty of content for players to look forward to. Today, exactly thirty days after the Kickstarter campaign began - and as it finally comes to a successful end - Sabotage Studio has thanked backers for their support promising that they will do their best to deliver the best product possible. Last month, Sabotage Studio announced that they were working on Sea of Stars - which is a role-playing game inspired by timeless classics like Chrono Trigger and the Final Fantasy series - and that they had just starter a Kickstarter campaign to help fund the project.įor those who may not be familiar with Sabotage Studio, it is the same developer that gave us the phenomenal retro-looking title The Messenger, and it even managed to win Best Debut Indie Game at the 2018 The Game Awards event very well-deserved, too. ![]() ![]() ![]() Presenter, “If I Knew Then, What I Know Now: Practical Advice for Aspiring Attorneys.” Western State University College of Law (Annual lecture 2012 – Present).“When Family Law Meets Criminal Law: Mastering Interplay Between Domestic Violence Litigation and Criminal Procedure.” Orange County Bar Association (September 2013) South Orange County Bar Association (June 2013).Moderator, “Beyond the Basics: An Expert’s Guide to Litigation of Complex Business Valuation and Cash Flow Issues.” Association of Certified Family Law Specialists – Orange County Chapter (November 2013).Presenter, “An Open House at 2640 Family Court: How to Properly Present Your Neighbors – the Epsteins, Watts, Moores, Marsdens, Nelsons, Brancos, Grinus and Dukes at Trial.” White, Zuckerman, Prego Program (May 2014).“Million Dollar Pre-Nup” Money Matters With Dino (November 2014).Presenter, “Divorce Cases from Start to Finish.” National Business Institute (November 2014).Presenter, “Substance Abuse Issues in Family Law Litigation.” Association of Certified Family Law Specialists, Orange County Chapter (November 2014).Presenter, “How to Comply with Subpoenas and Earning Assignments in Employee Proceedings.” Santa Ana Chamber of Commerce (April 2015).Presenter, “Protecting the Separate Property Interest (Or, would you rather go to the dentist?)”, First American Trust Family Law Seminar (May 2015).Panelist, “Crossover Issues in Trust & Estate Law and Family Law”, Orange County Bar Association & Trusts and Estates Section (October 2015).Presenter, “The Business of Law”, Whittier Law School, OCBA, Legal Aid Society of Orange County Boot Camp Training Event (February 2016).Presenter, “Protecting and Preserving Business Interests During a Marital Dissolution Action.” Orange County Bar Association Business and Corporate Section (March 2016).Presenter, “Divorcing the Company: A Family Law Practitioner’s Guide to Analyzing and Challenging Corporate Documents.” Orange County Bar Association Family Law Section (April 2016).Presenter, “Community Property and the Estate Planning Process.” First American Trust Family Law Conference (April 2016). ![]() Co-Presenter, “Preparing and Presenting Credits and Reimbursements Issues, Determining Lender’s Intent, Understanding the Accounting of the Right to Reimbursement Pursuant to Moore/Marsden and Beyond (Branco and Grinius).” Association of Certified Family Law Specialists – OC Chapter (April 2017).Co-Presenter, “Understanding and Winning Reasonable Compensation Issues.” OCBA Family Law Section (May 2017), White, Zuckerman, Prego Program (November 2018).Presenter, “Family Residence Accounting: Beyond the Numbers.” California Society of CPAs (January 2018).Presenter, “Date of Separation: Its Importance and Application from Petition through Judgment.” Association of Certified Family Law Specialists – OC Chapter (June 2018). ![]() Presenter, “What Every Criminal Defense Attorney Should Know About the Practice of Family Law.” OCBA Criminal Defense Section (Summer 2018). ![]() In his spare time, he stays active by running, and spending days at the beach with his wife, Susy, who is an attorney practicing immigration law. Schwartz as a presenter or panelist sharing his family law knowledge at some of Orange County’s most significant professional, legal and civic organizations. Schwartz has successfully litigated some of family law’s most complex multiple-day trials regarding child custody and visitation, domestic violence, child support and spousal support, attorney fees, characterization, valuation and division of community estate assets, valuation of business interests, as well as trials on the issues of the validity of a premarital agreement, transmutations, breach of fiduciary duty and date of separation. In addition, he has been a member of the Family Law Section of the Orange County Bar Association since 2001. Mr. He has been a Board Member on the State Board of Directors since 2014. He has also served as the organization’s Associate Director. In 2011, he became a member of the ACFLS, where he has served on the Executive Board of the Orange County section since 2012. He became a Certified Family Law Specialist in 2007. Schwartz has had an impressive career with the Association of Certified Family Law Specialists. ![]() ![]() ![]() ![]() Incompletely elongated DNA strands are presumably the consequence of the pausing of DNA polymerase on the template, or of its premature termination. Chimeric DNA molecules seem to result from the annealing of incompletely extended primers to a heterologous target sequence during PCR. Such PCR products complicate and can mislead the MPRA data analysis as well as decrease the productivity of the approach, as the association of the same BC with different ROIs leads to the elimination of all such BCs and ROIs from the analysis. However, it has been previously shown that routine conventional PCR co-amplification of DNA sequences containing two variable motifs (in the case of MPRAs, BC and ROI) separated by a constant region frequently leads to formation of undesired chimeric molecules, from 5.4 to 30%. To identify all unique BC–ROI combinations, PCR amplification followed by the next-generation sequencing (NGS) is typically used. Importantly, the sequences of BCs and/or the associated DNA regions of interest (hereafter ROIs) in MPRA plasmid libraries are frequently not known a priori, as random oligonucleotides are used to clone these elements. Thus, the influence of each DNA sequence on the reporter expression can be measured by counting of the associated BC in the reporter RNA-seq data. To trace each individual DNA sequence under study, each molecule in the library is uniquely marked by a short (from a few to up to several dozen bp in length) barcode (BC), which is present within an untranslated region of the reporter gene. ![]() Briefly, a set of DNA sequences to assay are cloned in a plasmid vector outside of a reporter gene coding sequence, and the resulting MPRA library, which typically contains a high number of plasmid variants, is used to transfect the cells of interest. enhancers and promoters) or their mutant variants. Massively parallel reporter assays (MPRAs) allow high-throughput functional analysis of different DNA regulatory elements (e.g. In addition, we found that there is a negligible difference in performance of emulsion and conventional PCR approaches performed with the identified settings. We have identified PCR parameters that ensure synthesis of specific (non-chimeric) products from highly diverse MPRA plasmid libraries. Using specific MPRA libraries as templates, we found that the two-round amplification of the BC–ROI regions with a very low initial template amount, an elongated extension step, and a specific number of PCR cycles result in as low as 0.30 and 0.32% of chimeric products for emulsion and conventional PCR approaches, respectively. To identify settings that minimize formation of chimeric products we tested a number of PCR amplification parameters, such as conventional and emulsion types of PCR, one- or two-round amplification strategies, amount of DNA template, number of PCR cycles, and the duration of the extension step. However, chimeric DNA molecules formed on templates with identical spacer fragment during the amplification process may substantially hamper the identification of genuine BC–ROI combinations, and as a result lower the performance of the assays. Typically, this is done by PCR amplification of the BC–ROI regions with flanking primers, followed by next-generation sequencing (NGS) of the products. In the latter case, it is necessary to identify these combinations before performing functional experiments. The sequences of BC–ROI combinations present in the libraries may be either known a priori or not. The assays are based on construction of highly diverse plasmid libraries containing two variable fragments, a region of interest (a sequence under study ROI) and a barcode (BC) used to uniquely tag each ROI, which are separated by a constant spacer sequence. Massively parallel reporter assays (MPRAs) enable high-throughput functional evaluation of various DNA regulatory elements and their mutant variants. ![]() |
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