Chris:LabNotes/FateMapping/Calendar/2015/2015-2-13
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C1 DNA-seq experiment (BA 41/42 2014_10_21 sort)[edit]
Background[edit]
- Previously ran a C1 DNA-seq on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-7-30>
- In order to obtain more samples, I'll be doing another C1 experiment, but this time using the same patient brain samples as Blue's RNA-seq project in order to have more relevant results to their project. The sample was taken from BA 41/42 2014_10_21 sort
Procedure[edit]
- For this experiment, I used the general protocol File:C1 DNA Seq protocol 100-7135.pdf
General procedure (from watching Blue load and run the IFC)[edit]
Prepping cells[edit]
- Thaw cells and really gently pipette nuclei with p20 (repeat pipette ~8 times)
- Use cell counter (if this is a new sort with unknown concentration of cells) = 8ul loaded onto haemocytometer (in the end, we want to load 600 cells onto the chip)
- Double check under the microscope that cells are intact in the haemocytometer.
- Normally would put in double amount/volume needed because if not all channels are filled/very low number of them are, we can just put the chip back on the C1 and press load again
- Make sure not clumping in haemocytometer in microscope. If the cells are clumped, just mix, let clumps settle to the bottem of the tube, then take only the top layer out to load
- Keep nuclei on ice until you have to use them
Priming IFC[edit]
- If you have nuclei you already know the concentration of, just leave them in -80C until actual priming starts then take them out to thaw
- Tap IFC to make sure all bubbles come to the surface and make sure all bubbles are out = if see any bubbles, just tap them with pipette tip to pop
- Make sure clean stage before putting on IFC
- Calculate for 600cells/5ul = make double (10ul) so just in case first load didn't work. In the example I watched Blue do, we mixed 1.8ul cells (at 671 cells/ul) + 5.2ul sort buff + 3ul C1 suspension buffer
- Load 9.5ul for chip (0.5ul to ensure you don't get a bubble)
- Gently mix nuclei then pipette out amount wanted then mix with sort/suspension buffer. Keep on ice until loading
Loading IFC[edit]
- Add cell wash buffer (no dye because no need staining)
- Add cells and go to C1 to load
Checking cells under microscope[edit]
- Make sure everything is on for the microscope (modules 1,2,3,4)
- Open up Micromanager 1.4 program (press okay when MCS popup window shows up)
- Open up C1 plugin ("C1 autocapture and processing" window) and perform the following steps:
1) Position initialization Chamber = 1 -> "set" (look at "live" in original micromanager program) (This will set the initial position 1) 2) Focus recorder (setting focus to all 4 corners = can only set focus if there is a cell in the capture site so move to the next closest well that contains cell) L Top -> 1 "Goto" "Get" (when in focus) L Bottom -> 46 "Goto" "Get" R Top -> 87 "Goto" "Get" R Bottom -> 96 "Goto" "Get" 3) Channels (keep as default) Brightfield Exposure = 8 Dapi Exposure = 20 Slice # = 3 Capture Range: Begin = 1 Z step = 10um End = 96 Output Folder = (You need to specify) = will output raw images into a "Source" subfolder then "Processed" subfolder will contain overlays 4) Before hitting acquire, move to 1st position (using "goto" for L Top) 5) "Acquire" = will take first the brightfield images then the DAPI
Some specific notes[edit]
- I loaded the following amount of cells to the medium size IFC:
1.5ul nuclei + 5.5ul sorting buff + 3ul C1 suspension buff
- Make sure to add 2mg BSA to the blocking reagent (otherwise, the loading will fail)
Results[edit]
- Below is the excel spreadsheet with the results from the picogreen assay