20.109(S14):Data analysis (Day7)

Introduction

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 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 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.

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? Does the table 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%.

First look at instructor mock/single/etc. samples plus own plots.

Then move to stats. Save-as Excel sheet, keep just what they need.

Prepare separate sheet with columns/calculations of interest.

Finally, copy in the data.

Add all B/G ratios and NHEJ ratios group Excel worksheet somehow?

For next time

Methods, as promised.

Reagent list

write something here or not accessible to edit

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