Notebook

Protocol for qPCR Validation of ChronoSeq results¶

Prepare 1000cells/μl of K562 Cells suspended in DMEM and load into the ChronoSeq device.
We will take Cell samples every 10minutes using the ChronoSeq device for 2 hours.
We will follow a protocol similar to SingleShot Cell Lysis Kit but we will not change our media to DPBS.
Prepare 12 PCR Tubes for Cell Lysis as follows and keep these Tubes on ice while your prepare. For each tube:
- Label the Tubes 1 through 12 for each timepoint.
- 48μl Single Shot Cell Lysis Buffer
- 1μl Proteinase K Solution
- 1μl DNase Solution
Start taking samples using the ChronoSeq device. Take 12 samples with a 10minute interval.
- Add 10ng/μl TNFα to the Cell Suspension after the first sample. Avoid using the same pipettes used for making Lysis solution.
- Adding 5 minutes after Schedule start is ideal.
- Record the time of addition with respect to the first sample.
For each tube:
- Add 5μl of Cell Suspension. Mix Well. This is equivalent to 5000 Cells.
- Keep the tube back on ice till you have collected all 12 Timepoints.
Spin down all the Tubes.
- Using a PCR Machine run the following program:
  - Lid Temperature 105°C
  - Volume 55μl
  - Incubate for 10 min at 21°C
  - Followed by 5 min at 37°C,
  - 5 min at 75°C.
  - 4°C for ∞
- The cell lysate can be stored on ice for up to 4 hr, at -20°C for up to 2 months, or at -80°C for up to 12 months.
- After PCR Immediately store the Tubes at -80°C

Adapted from existing protocol here.
The iTaq kit comes with ROX as a background reference dye so remember to set that in your qPCR machine. ROX is not necessary for Bio-Rad CFX Opus 96.
For each gene you will have to setup 12 qPCR reactions. For each 20μl reaction PCR Tube:
- iTaq™ Universal SYBR® Green reaction mix 2X: 10μl
- 1μl PrimePCR Assay primers (20X).
- iScript reverse transcriptase 0.25μl
- 4μl Cell Lysate.
- 4.75μl Nuclease Free Water
Setup the qPCR reaction as follows:
- 10min at 50°C for Reverse Transcription.
- 1min at 95°C for DNA denaturation and Polymerse activation.
- 40 Cycles of:
  - 95°C 10seconds Denaturation
  - 60°C 30seconds Annealing and Plate Read
Here is a screenshot of the plate setup
After the plate setup. The plate was sealed with a Optically clear adhesive film.
- Make sure the lips are completely sealed to avoid leaks. Then vortex the plate.
Plate was then spun down and loaded into the qPCR machine.
Run the Program.
Relevant files and results from Bio-Rad CFX Opus 96 can be found in this folder.
Here is an image of the Log_fold_change calculated from the results. A time delay for ICAM1 and TNFRSF9 can be seen.
You can look at the data analysis notebook here.
In the Gene Expression data it seems like TNFRSF9 is activated a little later compared to what we saw with the qPCR results.