20.109(S14):Data analysis (Day7)

Introduction

We hope that you’ll leave lab today with a sense of accomplishment, after inspecting your raw flow cytometry data and then calculating NHEJ repair values. Unfortunately or excitingly – depending on your perspective – it turns out in scientific research that the hard work is just beginning once the data is quantified! Interpreting the data and drawing (sometimes tentative) conclusions requires deep reading and thinking – a process that shouldn’t be rushed. To help get you started, let’s review a few key elements of DNA repair and of the NHEJ pathway in particular.

Our discussions of NHEJ have primarily been focused on Ku80 and DNA-PKcs, along with a few other key players such as Ligase IV, and will remain so here. However, the schematic below from the review by Grundy et al. highlights that many accessory players have their own roles to play. Recall that Ku80, with its dimer partner Ku70, completes the first step of NHEJ repair by binding to DNA double-strand breaks (DSBs). Keep this function in mind as you consider how a Ku80 knockout strain might respond to DNA damage. The Ku 80/70 dimer quickly recruits DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase, to form the DNA-PK complex. Although DNA-PKcs has some binding affinity for DSBs, this affinity is increased by two orders of magnitude by the presence of the Ku dimer, and the kinase activity itself cannot proceed in the absence of Ku. Interestingly, although the kinase activity of DNA-PK is known to be important, it is not certain precisely which phosphorylation events are absolutely required for NHEJ.

NHEJ pathway overview from Grundy et al. FINISH CITATION LATER

To better understand how DNA-PKcs may differ from Ku80 as a target for changing a cell’s DNA damage response, let’s take a closer look at Compound 401. A number of small molecule inhibitors of DNA-PK were discovered by Griffin et al. in the early 2000s [LINK]. While both Griffin and the commercial vendor Tocris/R&D Systems refer to the inhibitors generically as DNA-PK rather than DNA-PKcs inhibitors per se, it is clear that the "cs" is implied and kinase activity is what is being inhibited. The inhibitor is described as “ATP-competitive,” indicating that it competes for the ATP binding pocket in the kinase domain of DNA-PK. Moreover, the inhibition assay used p53 phosphorylation as a readout, rather than phosphorylation after binding to Ku 70/80. Finally, the inhibitor also acts on the unrelated kinase mammalian target of rapamycin (mTOR).

Topic 1: document some of the lecture info on NHEJ key players, leading to

Topic 2: more about C401 inhibitor, and

Topic 3: more about colony-forming assay and staining approach

Topic 4: but the most interesting/fun will be flow analysis: mean vs median choice; breaking down the Day 5 equation a bit more

Oh, and just a word about the MCS/GC/etc. issue for context

Protocols

Part 1: Stain irradiated cell colonies

Option to do it on M3D1 if they want to grow longer for bigger colonies?

All in main lab: rinse w/ 2mL pre-warmed PBS, add 2 mL Coomassie for 1 hr w/shaking, save it afterward, rinse w/PBS again, let dry a little bit, then manually count colonies right away. (Suggestions for counting approach and how to decide which ones pass the threshold.)

Part 2: Flow cytometry analysis

Overview:

• You will begin by looking at images from the instructor samples to learn how to read the flow cytometry plots and summary statistics.
• Next you will peek at your own images and form preliminary expectations about your data set.
• Finally, you will work in Excel to precisely calculate the NHEJ repair value for each of your three conditions (two replicates each).

Protocol:

1. On one of the lab computers, double-click on the FACS server shortcut.
   • Alternatively, on your own computer access 18.159.2.11 directly. Ask your instructors for the username and password.
2. Go to the April 2014 folder, then to Agi Stachowiak. Copy over both the T/R and W/F image sets to your laptop: the filenames begin "analysis-images" and only the dates differ.
3. Copy over just your own day of statistics, unless you really want access to all of the raw data in your back pocket: the .csv filenames begin "analysis-statistics" and only the dates differ.
4. The instructor samples are listed in the table below. From this table, and from the T/R and W/F image sets, try to address the questions below.
   • Background. The scatter data is used – in three steps – to make gate P3, which should consist primarily of live, single cells. From the cells gated in P3, two sub-gates are made that capture all GFP-positive cells ("Green cells" gate) and all BFP-positive cells ("Blue cells" gate). Both singly and doubly positive cells are included in each gate. It is important to read the "% Parent" statistics: these indicate XFP-positive cells as a percentage of all the cells in P3. The "% Total" statistics include debris, aggregates, and clearly dead cells!
   • What percent Green cells are in the mock sample on each day? What about Blue cells?
   • What percent of singly-transfected cells express GFP? Do within-day and cross-day replicates agree well or not?
   • What percent of singly-transfected cells express BFP? Do within-day and cross-day replicates agree well or not?
   • What percent of co-transfected cells express GFP? Express BFP? Comparing the Green and Blue gates to Q1 and Q4, about what percent of cells seem co-transfected, versus expressing just GFP, and expressing just BFP?
   • How is within-day and cross-day replicate agreement for the co-transfected samples? Do the tables below suggest an explanation for why?
   • Does ethanol appear to affect scatter profiles? What about affecting GFP, BFP, or co-expression?
   • What NHEJ repair value do you calculate for Zac's original BFP plasmid, using the first replicate in the W/F instructor data? Try this calculation by hand, using the mean fluorescence intensity. Later, you can include this data as a check on your Excel worksheet. The value you should calculate is 12.8%.
5. After you understand the instructor data, skim over your 12 sample plots. Can you see apparent differences between K1, K1+401, and xrs6?
6. Now that you have a good conceptual understanding of the data, it's time to crunch some numbers. Open the .csv file and save it as a newly named .xlsx file.
7. Begin by deleting all of the rows except the twelve containing your own dataset.
8. Next delete all of the columns except the few that interest you. Keep in mind that you need to know Green cell and Blue cell gating as a % of the parent gate, P3. Class-wide, you are only required to do your calculations based on mean fluorescence intensity (MFI), to be consistent with Samson lab data. However, you may find it interesting to see whether using median fluorescence intensity gives you the same trends or not. Just a few extra copy-pastes to do both calculations!
9. We recommend that you prepare a new Excel file with your NHEJ equations, and just copy-paste in the appropriate % and MFI data; this approach is a versatile one. Your final worksheet might look similar to the screenshot below.
10. Remember that for each of the twelve wells you should calculate raw reporter expressions and a BFP/GFP normalized value. Then, for each intact/cut pair you can calculate an NHEJ value. In this way, we should have quadruplicate NHEJ values for most repair topology/cell population conditions, which will allow us to do statistical comparisons.

Reference information:

TR instructor samples.

WF instructor samples.

Sample NHEJ calculator screenshot.

You must email your Excel sheet to 20109 DOT submit AT gmail DOT com before leaving lab today. We instructors will post a summary file for ease of class-wide data analysis by Wednesday evening or Thursday morning.

For next time

Recall from last time:

1. Revise your earlier draft of the Methods section, just through M2D2, applying the feedback you received.
2. Prepare the rest of your Methods section (through M2D7) in outline form. Start by considering what methods may be logically grouped together. At a minimum, you should turn in
   • sub-section titles,
   • topic sentences for each sub-section,
   • and a few short phrases indicating what content will be included in that sub-section.
     • The phrases do not need to include every single material/concentration/etc. that you will use, but they should convey the scope of that information in very abbreviated form.
     • For example, phrases for the first half of a Western sub-section could look like: cell lysis method (RIPA and inhibitors from BBP, scraping, ice 15 min and spin 15 min); Precision Red to measure protein in supernatant; preparation step (add Laemlli, boil equal amounts of protein). The second half would include similar types of phrases to cover the PAGE and transfer steps.

Reagent list

• PBS
• Bio-Safe Coomassie Stain (Bio-Rad)
• Mostly your brains!

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