![]() ![]() They don’t do that at all anymore, which certainly makes me happy."I would recommend Oncue to other moving companies. Before, people were complaining a bit about things being slow. “The biggest change with our new infrastructure is that, in general, our network runs much better. They also have the peace of mind knowing that any updates they might have to make will be far less expensive than the inevitable refresh they would face if they had opted for a more traditional storage solution. The Nutanix hyperconverged infrastructure actually gives them more flexibility and scalability than ever before, ensuring that they can continue to grow without having to worry about outpacing their infrastructure. As if those perks weren’t enough, their new infrastructure isn’t merely designed to help them meet current needs. In the end, vastly improved performance, increased storage capacity and an easier administrative interface have made operations at OnCue much smoother. Since OnCue went with a hyperconverged infrastructure, they’ve already seen numerous additional benefits. But that could be achieved with any new storage solution. ![]() First, more storage was added, giving OnCue the ability to keep growing without worrying about free space. – Amanda Taylor, Systems Administrator, OnCue Express The Difference Hyperconvergence Makesįor OnCue, hyperconvergence turned out to be as advertised. They helped to streamline the whole project, making it a top priority.” “Doing the implementation by ourselves would’ve taken three times longer without InterWorks. These controllers also handle moving VMs in the background, removing the need to do that manually. ![]() The best part of configuring this new system is that the need for ongoing maintenance is greatly reduced thanks to each host orchestrating updates through controller VMs. InterWorks assisted in this configuration process, getting the solution up and running quickly and with minimal interruption to OnCue’s operations. With Dell doing a lot of the heavy lifting in delivering the three pre-provisioned hosts with Nutanix and ESXi installed, all that remained for OnCue was configuration. Even better, Nutanix would run on Dell XC730 hardware, in keeping with OnCue’s affinity and experience with Dell products throughout their infrastructure. After examining all the options closely, InterWorks and OnCue agreed that a hyperconverged solution from Nutanix fit both the requirements and the budget over the competition. To begin, InterWorks helped OnCue identify a few hypeconverged infrastructure options. No stranger to the revolutionary world of hyperconvergence, InterWorks agreed that hyperconvergence was indeed an excellent fit for OnCue. Which hyperconverged solution would be the best for their needs? How would it fit in with the rest of their IT infrastructure? To guide them in this decision-making process, they turned to their longtime IT partners at InterWorks. The Decision for HyperconvergenceĪny new technology comes with a degree of uncertainty. But as operations have continued to intensify, OnCue has taken a more aggressive approach in anticipating their future needs. In fact, the EqualLogic storage they had been using lasted quite some time. That’s not to say that their older system didn’t serve them well. They knew they needed equipment that could not only keep up with the current pace of growth but scale out with them as they grow in the future. As they continued to expand across Oklahoma, they knew it was only a matter of time before their more traditional, 3-2-1 IT infrastructure could no longer meet their needs. OnCue has enjoyed steady business throughout their 50 years, but their growth has skyrocketed over the past decade. With meticulously maintained facilities, customizable fountain drinks and an exhaustive selection of foods that goes beyond just snacks, OnCue’s stores aim to be the ideal one-stop convenience location for its customers. In addition to serving up quality fuel, OnCue strives to make their stores premier convenience locations. Founded in 1966, OnCue provides Phillips 66 Top Tier Gasoline to its customers while also pioneering compressed natural gas (CNG) as an alternative domestic fuel. OnCue is an Oklahoma-based convenience store chain with over 65 locations across the state. OnCue is a convenient store chain based in Stillwater, Oklahoma, with locations across the state. ![]()
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![]() If you make additional changes to your model that you want to include in this migration, then you can re-scaffold it by running 'Add-Migration InitialModel' again. This snapshot is used to calculate the changes to your model when you scaffold the next migration. The Designer Code for this migration file includes a snapshot of your current Code First model. However, if I skip the enamble-migrations command and simply run the application the database is successfully created (but, of course, no Migrations directory in the project directory).ĭoes anybody know how to solve this issue so that I can continue the Code-First part of the course?Īfter I de-installed SQL Server 2019 Developer and installed SQL Server Express I could run the following commands successfully: PM> enable-migrationsĬode First Migrations enabled for project CF_NewDB. The line “Checking if the context targets an existing database…” takes a while and the path should have been created by the enable-migrations command. I believe the “Could not find file” error is a red herring. Using VS Community 2019 and SQL Server 2019 Developer. My connection string in App.config (which I took from the DatabaseFirstDemo App.config file): System.IO.FileNotFoundException: Could not find file 'C:\Users\Arjen\Source\Repos\CodeFirstDemo\Migrations'.įile name: 'C:\Users\Arjen\Source\Repos\CodeFirstDemo\Migrations'Īt System.IO._Error.WinIOError(Int32 errorCode, String maybeFullPath)Īt System.IO.Directory.InternalCreateDirectory(String fullPath, String path, Object dirSecurityObj, Boolean checkHost)Īt System.IO.Directory.InternalCreateDirectoryHelper(String path, Boolean checkHost)Īt .(ScaffoldedMigration scaffoldedMigration, Boolean rescaffolding, Boolean force, String name)Īt .()Īt .CommandLineApplication.Execute(String args)Īt .Program.Main(String args)Ĭould not find file 'C:\Users\MyName\Source\Repos\CodeFirstDemo\Migrations'. ![]() ![]() When following the video “ Demo -Code-first Workflow” I got the following error when running enable-migrations in the Package Manager Console: PM> enable-migrationsĬhecking if the context targets an existing database. ![]() ![]() ![]() After completing, to the toolbar, then pick” Produce ISO” to build a setup image. That is your options are displayed around the left aspect of this NTLite window. Once that is finished, you should begin to make alterations in Windows. Currently, the next slow-loading process can begin. Click on it together with the most suitable mouse, and pick” Export”. And it is preferable to make use of NTLite. And with the preferred Windows identity (offer a taste of this downloading period into 10). However, when you end, you’ll possess the thing together. ![]() NTLite Crack app might have a couple of minutes to approach the ad. This is a tool that permits one to personalize ISO graphics to put in Windows on your computer with a few essential alterations. But special to the ingredients of your PC. Further an upgrade to it and operators not located in the primary image. After it is conceivable to use many Windows peculiarities in the disc model. This is deservingly computing that you can edit choices compared to preserving the most important factors and system organizers. You can get the benefit of this application to monitor image files in Windows. Now, when you’re using this software, you can make adjustments to Windows that are currently working on your personal computer, and you don’t have to put the process of rebooting your PC into those windows that are edited, and after it’s finished. This is compatible with 64-bit and 32-bit versions of Windows. NTLite Crack is an excellent application that can build custom picture files for Windows. Download Setup NTLite 20 Crack With Keygen ![]() ![]() ![]() Hyper-V Manager – Managing individual Hyper-V hosts, creating virtual switches.What are the GUI tools that administrators typically make use of with Hyper-V? These include: Like other hypervisors, Hyper-V provides the ability to carry out needed tasks in GUI tools that can be used with Hyper-V. Managing, configuring, and administering Windows Server Hyper-V on a day-to-day basis includes performing many different tasks. PowerShell’s command-line capabilities provide quick and easy ways to accomplish many tasks and actions in a much more efficient and effortless way than using the GUI-based counterparts. PowerShell provides a very “human-readable” verb-noun construct that is relatively intuitive and easy to learn, even for beginners.Įven outside the Microsoft ecosystem, most solutions today provide PowerShell integration to provide easy automation and configuration. NET Core is the underlying object-oriented language. ![]() ![]() What is PowerShell?įor those who have not heard of or used Microsoft PowerShell, it is an object-oriented scripting language built on top of. This post will feature the top 10 PowerShell commands for Hyper-V, why they are useful, and how to use them. ![]() Combining Hyper-V and PowerShell features allows IT administrators to have the features and capabilities needed to configure, manage, and administer their Hyper-V environment. NET framework provides a fully object-oriented underlay that makes the language extremely robust. PowerShell is a relatively straightforward scripting language built on top of. One of the powerful features offered by Hyper-V is management with PowerShell. Hyper-V contains relative feature parity with other hypervisors on the market and allows businesses to maintain alignment with current Windows Server technologies they are already using on-premises and in the cloud. I hope this was helpful, and if you have any questions, feel free to leave a comment.Microsoft’s Hyper-V hypervisor is an extremely powerful enterprise-ready hypervisor that has grown and matured in recent Windows Server versions. If you are running PowerShell 7, there is a cross-platform cmdlet called Test-Connection, which provides you with similar capabilities. Test-NetConnection is part of Windows PowerShell 5.1 and is available on Windows Server as well as on Windows 10. If you want to learn more about the Test-NetConnection cmdlet to troubleshoot network connectivity issues, check out Microsoft Docs. Test-NetConnection -ComputerName -DiagnoseRouting -InformationLevel Detailed You can also perform route diagnostics to connect to a remote host. Test-NetConnection -Port 443 -InformationLevel "Detailed" However, there is more! One of the commands I need the most, especially when working with web services, is to test a specific TCP port. Test-NetConnection -InformationLevel "Detailed" You can do simple things, like just testing ping connectivity:īut you can also get some more detailed information on the connectivity: Depending on the input parameters, the output can include the DNS lookup results, a list of IP interfaces, IPsec rules, route/source address selection results, and/or confirmation of connection establishment. It supports ping test, TCP test, route tracing, and route selection diagnostics. The Test-NetConnection cmdlet displays diagnostic information for a connection. My favorite PowerShell cmdlet to troubleshoot network connectivity issues is Test-NetConnection. Luckily, Windows Server comes with PowerShell and has build-in cmdlets to help with that. So as a server admin, we need to have a tool to troubleshoot network connectivity issues on Windows Server to figure out is DNS working, is the remote endpoint even reachable, is the port open, and many other things. We all have been there, we want to set up a new application or agent which needs network access and needs to reach an endpoint using a specific port, but it simply doesn’t work. See the original author and article here. ![]() ![]() ![]() See also: When Practicing Archery What Hand Do You Hold The Bow With? (Perfect answer) Does Tamu have a gym? There are four basketball courts, four volleyball courts, and twelve badminton courts available in the gym. There are currently three gymnasiums available for use for basketball, volleyball, indoor soccer, and badminton, as well as other sports. ![]() Visit for the most up-to-date information, resources, and prevention measures about COVID-19. While face covers are recommended, they are not essential. The Student Recreation Center and Polo Road Recreation Center are both fully operational and fully occupied. – 10:00 p.m Beginning on Monday, March 16 and continuing through on Saturday, March 21 from 8:00 a.m. to 4:00 p.m., Monday, March 16 – Monday, May 25, except holidays. Neither a member of the Blinn team, nor a regular old blinn student. The Member Services desk (97) is available to assist guests with membership sales, locker rentals, lost and found, and other general inquiries. Male and female locker facilities with approximately 2,300 lockers in three sizes, as well as private shower spaces, hair dryers, and vanity areas with power are available in both buildings. It is available throughout all operation hours and offers our members and guests a convenient option to the main entrance. A handy alternative to the main entry, this entrance is located on the southwest side of the Rec Center near Olsen Field and adjacent parking lots. ![]() ![]() ![]() ![]() ![]() Download and use Farbar Recovery Scan Tool for free. Farbar Recovery Scan Tool, or FRST, is a portable application designed to run on Windows XP, Windows Vista, & Windows 7/8/10 in normal or safe mode to diagnose malware issues. When the scan is complete, the program saves the finished report in the same folder where the executable file is stored. Farbar Recovery Scan Tool 64-Bit has been released, as well MezerTools 2.3.4.0, foobar2000 1.6.7 Beta 4, CleanDNS 5.144.21.1, Shopbook 4.60, Biniware Run. After analyzing the report, forum regulars usually provide a file with edits (Fixlist), which you can apply on your PC at your own risk.įarbar Recovery Scan Tool is a portable program and can be launched from removable media. Farbar Recovery Scan Tool 64-Bit Farbar Recovery Scan Tool, or FRST, is a free portable application designed to diagnose malware issues. You can download FRST from one of the following links (the files will be downloaded from ), and then follow the instructions below to run it. If you've ever attended an online anti-malware forum, you're sure to know that when dealing with a problem, experienced users most often require reports created by Farbar Recovery Scan Tool and other specialized programs (such as AVZ). FRST (Farbar Recovery Scan Tool) is a free third-party tool that can be used for diagnostics and malware removal. Typically, such programs are used to provide remote assistance in cleaning the system from contamination. Welcome to the download page for O&O SafeErase Professional 圆4, a top-tier Windows 10 software developed by the renowned O&O Software GmbH. Advanced users can create a file of edits on their own and "feed" it to Farbar Recovery Scan Tool. The user will have to fix most of the found problems on his own, this tool can only undo changes made to some system files and make several other corrections. It can analyze your Windows system and compile text. The program generates detailed reports on all processes and files, helping to identify the presence of malware on the user's computer. Farbar Recovery Scan Tool can fix problems that are commonly caused by malware infections in Windows. Farbar Recovery Scan Tool, or FRST, is a portable application designed to run on Windows XP, Windows Vista, and Windows 7/8/10/11 in normal or safe mode to. Farbar Recovery Scan Tool is a portable tool that can collect detailed information about the system and the activity of various programs/services. ![]() ![]() ![]() Somewhat less conspicuously, it is also one of the few games that can accomodate multiple monitors in a useful way.Supreme Commander has much greater multi-core support than most games, with performance scaling heavily with the number of cores.Cryostasis had extremely advanced fluid simulation on release, although even recent computers can have trouble running the game thanks to a lack of multi-core processor support.It is arguably one of the first applications that will have a real impact in driving people to upgrade from Windows XP to Windows 7. This means it is not playable on Windows XP machines. Battlefield 3 has no ability to use Direct X 9.The trouble is, RAM was expensive back then, and most of the world was fine on 256MB. In order to take advantage of the large draw distance (which was unheard of at the time), one needed 1GB for maximum draw distance. Later patches added Shader Model 3.0 features like HDR lighting that could only be utilized by the then-newly-released GeForce 6800 cards. Far Cry was actually a tech demo first for NVIDIA's GeForce 3 cards.CryTek almost averted this with Crysis 2, until they released a patch that enabled Direct X 11 functions and all new "Ultra Detail" mode.SCP Foundation -062 is a highly advanced quantum computer, believed to be from the future. ![]() This had the unforeseen additional effect of lots of people pirating the game just to use it as a tech demo, just because they "didn't want to pay for the whole thing" (and didn't think to check if Crytek had released a demo version).Computer Shopper still uses it to bench-test new hardware. Although the lower settings could perform well and still look better than most games at the time. Yahtzee summarized it best, saying that the game must have been designed for some ultra high-tech supercomputer from space. Even fewer could run the "Very High" setting at 1280x800 above 10FPS. When released, only a handful of computers could actually handle the "High" setting at 1280x800. Virtua Racing, Sega's first 3D arcade game, was originally designed as a proof-of-concept demo for the state-of-the-art polygonal graphics hardware which (with some upgrades) would go on to produce such hits as Virtua Fighter and Daytona USA.The 3D board it featured was actually quite huge, looking like several modern-day computer motherboards stacked. Hard Drivin is seemingly the first 3D racing game.I, Robot provided a demonstration of 3D raster graphics.Note that this trope has nothing to do with the respective quality of a game in itself, and many games started with the motivation of being a tech demo end up as extremely well received classics for much more than just their technical aspects. In the PC gaming world, this becomes a problem as the game expects nothing less than bleeding edge hardware. In a video game console setting, this isn't such a problem unless the hardware in question is the latest peripheral. ![]() The problem though is that most of the time the technology they're trying to show off is hardware based, which limits who can actually play it. But this isn't unlike an Obvious Beta, the developer tried in earnest to make a fully functional game that plays relatively well. It can be loosely described as a tech demo disguised as a game. This is where the Tech Demo Game comes in. Or in an environment that's limiting such as video game systems, smartphones, and tablets, a proof of concept to show that hey, that piece of hardware really can do it.Īnd what a better way to show off the technology by having users interact with it in a fun way. This usually happens in the PC hardware world where graphics card vendors put out tech demos to show it off. Whenever some new technology comes out, companies want to find ways to market their product. PAGES WILL BE DELETED OTHERWISE IF THEY ARE MISSING BASIC MARKUP. DON'T MAKE PAGES MANUALLY UNLESS A TEMPLATE IS BROKEN, AND REPORT IT THAT IS THE CASE. THIS SHOULD BE WORKING NOW, REPORT ANY ISSUES TO Janna2000, SelfCloak or RRabbit42. The Trope workshop specific templates can then be removed and it will be regarded as a regular trope page after being moved to the Main namespace.
![]() ![]() Added an icon on Firewall rule in order to indicate that Firewall Logging is enabled Added the ability to use an external TCP Accelerator to support the WAN connections Added web filtering categories to Outbound Policy for SpeedFusion / PepVPN Sub-Tunnel Custom DNS Servers in LAN DHCP Server can now be pushed to client Added the ability to push DHCP Option 15 to client Added the ability to define port number and to choose whether to use UDP or TCP mode Added ability to push WINS server directly to clients Added the ability to define the Allowed WAN Interface for SNMP under System > WAN Connection Access Settings Updated Daylight Saving Time support for "Pacific/Fiji" and "Australia/Lord_Howe" regions ![]() Support TACACS+ for web admin access authentication IP Forwarding WAN supports NAT for Multiple LAN network Added UDP protocol supporting for Custom Service Forwarding Added the ability to access CLI from the WAN via LAN/VLAN IP in IP Forwarding mode Added support of telnet command inside SSH CLI Added support for ECDSA certificates in the Certificate Manager Updated supported browsers: Mozilla Firefox 54, Google Chrome 51, Safari 10, Microsoft Edge 15 The Web UI now uses the "Device Name" instead of the "Router Name" on the Status and System pages The Firewall Log will now put in its own page rather than in the Event Log After pressing Apply Changes, the WebUI will now stay on the same page and display a message instead of going to the Dashboard page Added the Ability to access Web Admin page from IP Forwarding WAN via LAN/VLAN IP Added a TB scale in addition to MB, GB in Usage Reports ![]() Added support for using the default LAN IP address as the outgoing IP for Local Services Added support for UDP tests of up to 2 Gbps bandwidth If Server/Port information is not specified for URL or Session Logging, Remote Syslog server settings will be used URL Logging has been relocated from Content Blocking page to System > Event Log. Updated the protocols of DPI Report which will be consistent with Web UI Added the ability to detect P2P, Gaming categories, and IoT Protocols Fixed an issue where the DHCP server may not reply to DHCP discovery or requests in rare situations Added ability to disconnect the Wi-Fi and Remote User Access clients The CLI now uses the"Device Name" instead of the "Router Name" when responding to the "get system" command. Added support for performing ping / traceroute from specific LAN or WAN Interfaces The Status display now shows exported routes Added the ability to set a new Billing Cycle Added the ability to send USSD codes via cellular connections ![]() An Icon will be displayed on Cellular status of Dashboard when Bandwidth Allowance Monitor usage hits 100% and "Reserve for management traffic" option is enabled Added support for prioritizing cellular carriers Added custom hosts for ping target on SmartCheck Added Support for 16 Captive Portal Profiles in enterprise-grade modelsĬompatible with Balance: 305/380 or above Added the ability to receive SMS messages through cellular connectionsĬompatible with Balance: 20X, 30 LTE/Pro, 310X Added support for 802.11k neighbor report base on Wireless SSIDs Added support for WPA3 security policyĬompatible with MAX: BR1 MK2, Transit, 700 HW3 or above, HD2 HW5 or above, HD4 Added support for GPS time as time sync source and able to select GPS as the primary time sync source under System > TimeĬompatible with MAX: BR1/2, HD2/4, Transit Added NetFlow Support (To enable this feature, please go to Compatible with Balance: 30 LTE/Pro, One/Two, 210/310 HW4 or above, 305/380 or aboveĬompatible with MAX: BR1 ENT, Transit, 700 HW3 or above, HD2 HW5 or above, HD4Ĭompatible with X Series: EPX, SDX, MBX, 310X, 20X Added support for NTP ServerĬompatible with MAX: BR1 ENT, 700 HW3, HD2/4, Transit Revamped SpeedFusion Chart shows additional statisticsĬompatible with X Series: EPX, SDX, MBX, 310X (To enable this feature, please go to and choose Traffic Distribution Policy) Added Dynamic Weighted Bonding: a new SpeedFusion bonding algorithm for dynamic bandwidth connections, eg: cellular. Wi-Fi clients connect to the newly created SSID will use the related SpeedFusion Cloud link for Internet accessĬompatible with MAX: 700, BR1/2, HD2/4, Transit (Exclude: BR1 ENT, HD2 Mini, HD1/2 Dome) Added ability to create the new SSID for the SpeedFusion Cloud profile under the new SpeedFusion Cloud page. Added Dynamic Weighted Bonding algorithm support for SpeedFusion Cloud (To enable this feature, please go to and choose Traffic Distribution Policy)Ĭompatible with All Models (Except FusionHub) Added ability to select the LAN device to route all traffic to the SpeedFusion Cloud link for Internet access Added ability to connect up to three region of SpeedFusion Cloud ![]() ![]() ![]() There are ways to work around this, but it's just a bit more friction in my daily work. Paths are different and there is no proper support for rpm-ostree package installations in Ansible, for example. AutomationĪutomating Silverblue has also some issues. Rpm repositories are chosen by default here and there, and it just feels a tad more sluggish than in Fedora Workstation.įedora Workstation on the other hand has very well maintained packages, but misses the cool rollback feature from ostree. Update notifications are missing very often. It has really improved in version 40, but not on Silverblue. Not sure where it is coming from and why it happens, but it's there. A randomly crashing extension server in vscodium is such an example. Furthermore, some flatpak applications seem very unreliable. how can I get rid of the flatpak internal nautilus file picker". This does not make the system itself unreliable, but the behavior of different applications can vary from "works like a charm" to "phew. Many messengers want to read and write files to your home directory. An editor or IDE that wants to use ShellCheck for example needs special treatment in most cases. Some software may need access to system tools. Some flatpaks don't have permissions to use your home directory, for example. This is a nice idea, but introduces some hurdles. Due to the nature of Fedora Silverblue, you will have many applications coming as flatpak or put into a container or toolbox. But there are some things, you need to consider. I don't care if I have to reboot a machine occasionally or dig into some issues. Silverblue and Workstation are working very reliable for me. So, I am not switching due to the lack of documentation or a smaller community. In the end, you will have basically the same situation and getting answers for most topics. On the community support side, you can expect that the Fedora Workstation community is way bigger, but most people working with Silverblue are deeper in the ecosystem. The team is small and tries its best, the documentation is not as up-to-date as you might desire, sometimes. Some additional, but very limited, documentation is available. In some regards, you can use documentations for Fedora Workstation and apply them to Silverblue. Support & Documentationįrom the support and documentation point, both are very different. My decisionīefore digging into more details: With Fedora 35, I will switch back to Fedora Workstation. I wanted to write a dedicated article about all the benefits and features of Fedora Silverblue. In fact, I was so amazed, that I used Fedora Silverblue for over 2 years on production machines at work and home. Overall, it is an amazing vision to have an immutable base OS and sandbox/container applications on top of it. Most of the tooling is still Fedora Workstation alike - You are getting Gnome 40, Firewalld, SELinux and more. ![]() The recommended way to deploy applications and services is via Flatpak, Podman or Toolbox (a somewhat special Podman container). For example, you are getting an immutable disk layout, which can be updated and manipulated via ostree/ rpm-ostree.įedora Silverblue is also very interesting, since it heavily focuses on containers. It adds some interesting twists to the "good ol' GNU/Linux". ![]() Screenshot - Notes & ToDo Fedora Silverblueįedora Silverblue is a quite new variant of Fedora. I have written a more detailed article about Fedora Workstation and how to automate it, already. That does not mean the others are irrelevant, but for me, it was the best bet for a long time. It is a bit like "current as Arch Linux, but comfortable as Ubuntu". With Gnome 40, Firewalld, SELinux, Podman and very up-to-date software it is one of the best distributions I know of. CentOS on production servers, Red Hat Enterprise Linux on highly critical machines and Fedora on the desktop or development machines. Using Fedora Workstation is something I was doing for a very long time (8 years or so) in production at work and home. ![]() But what are the differences? Why have I chosen to use Fedora Workstation after two years of Fedora Silverblue? Fedora Workstation With Fedora Silverblue, you are getting the option to dip your tows into immutable desktop distributions. The Fedora project provides many flavors of Linux based Desktop operating systems. ![]() ![]() Error bars represent SEM of three biological replicates. Right: Quantification of H3 K36me 3 signal after normalizing to total H3 signal. Extract from a set2∆ strain was used to confirm specificity of the H3 K36me 3 antibody. Library plasmids were transformed into KY812 for plasmid shuffling. ( A) Left: western blot analysis of H3 K36me 3 levels in H3 and H4 mutant strains. All heatmaps and metagene plots were generated using deepTools2 ( Ramírez et al., 2014 Ramírez et al., 2016) using 25 bp bins. Arrows above gene names indicate directionality of transcription. IGV-visualized BAM-file snapshots depict presence or absence of strand-specific read-pairs between genes. Regions were chosen to be void of neighboring ncRNA loci. Data represent log-scaled, spike-in normalized 4tU-seq read density over the plus (+) and minus (-) strands. ( C) Browser tracks visualized in IGV ( Thorvaldsdóttir et al., 2013) depicting aberrant transcription 5’ and 3’ to divergent (top), convergent (middle), and tandem (bottom) gene pairs in the indicated strains. ( B) Metagene plots show averaged intensity over regions displayed in the difference heatmaps shown in panel A. Heatmap rows represent 1186 divergent, 1981 convergent, and 2976 tandem protein-coding genes showing 500 bp upstream of the +1 nucleosome ( Brogaard et al., 2012) and 500 bp downstream of the CPS ( Ozsolak et al., 2010). ( A) Heatmaps showing log2-fold change in spike-in normalized 4tU-seq read counts in H3 R52A or H3 T45A mutants relative to WT at divergent, convergent, and tandem genes. All heatmaps and metagene plots were generated with deepTools2 ( Ramírez et al., 2014 Ramírez et al., 2016) using 25 bp bins. Antisense heatmaps are the reverse of the sense heatmaps plotting (from left to right on the x-axis) the region from 500 bp upstream of the CPS to 500 bp downstream of the +1 nucleosome (dotted line) on the non-coding strand. ( C) Expression of regions antisense to the 6205 protein-coding genes shown in Figure 3A in the H3 T45A and H3 R52A mutants compared to WT. Heatmaps plot the region 500 bp upstream of the +1 nucleosome to 500 bp downstream of the CPS (dotted line) and are sorted by gene length. ( B) Heatmaps sorted by gene length and showing spike-in normalized FLAG-Rpb3 ChIP-seq read counts (gray scale) and log2-fold change between the H3 R52A mutant and WT at 6205 protein-coding genes. Box and whisker plots were generated in R Studio ( RStudio Team, 2016) and p-values were calculated using the Wilcoxon-Ranked sum test (***p<0.001). ( A) Box plots comparing log2-transformed spike-in normalized RNA-seq read counts in a window 150 bp downstream of the CPS. Structure from PDB ID 1ID3 ( Luger et al., 1997). H2A, H2B, H3, and H4 are colored in pink, yellow, lilac, and green, respectively. Due to its buried location, H3 Q93 is not marked. ( E) X-ray crystal structure of the nucleosome denoting histone residues (highlighted in red) identified in the termination reporter screen and through northern analysis. The northern blots are representative of three independent experiments. Arrows correspond to those shown in the locus diagrams in panel C. ( D, F) Northern blot analysis to assess transcription readthrough of SNR genes in ( D) H3 and H4 mutants (plasmids shuffled into KY812) and ( F) H2A mutants (plasmids shuffled into KY943). For SNR13, the probe overlaps the downstream gene, TRS31, and detects a readthrough transcript of SNR13 (black), as well as the full-length TRS31 transcript (green). The intergenic SNR48 probe detects a single readthrough transcript. The intergenic SNR47 probe detects two read-through transcripts, as indicated by the long black and short blue arrows. The black bar over each locus denotes the probe position. ( C) Diagrams of three snoRNA loci analyzed for termination readthrough by northern analysis. For each strain, a 10-fold dilution series (starting at 1 × 10 8 cells/mL) was plated to SC-Trp as a growth control and to SC-His-Trp + 0.5 mM 3-aminotriazole (3-AT), a competitive inhibitor of the HIS3 gene product, to identify mutants expressing the HIS3 gene. Library plasmids ( TRP1-marked, CEN/ARS) ( Nakanishi et al., 2008) expressing the histone gene mutations were introduced by plasmid shuffling into strains expressing the SNR47 termination reporter (KY3220 left) or the reporter control (KY3219 right). ( B) Yeast dilution assays to monitor growth of strains expressing the indicated H3 and H4 derivatives as the only source of H3 or H4. Black arrows denote the transcripts produced from each reporter in wild type (WT) and termination mutant backgrounds. ( A) Yeast strains contain either an SNR47 transcription termination reporter (top, KY3220) or a control transcription cassette lacking the SNR47 terminator (bottom, KY3219) integrated at the LEU2 locus. ![]() |
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