Alice:Single/2015-10-23: Difference between revisions
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>Zsakura2 |
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*prepared the C1 reaction according to manufacture protocol: | *prepared the C1 reaction according to manufacture protocol: | ||
*Picogreen quantification | *Picogreen quantification | ||
{| {{table}} | {| {{table}} border = 1 | ||
| align="center" style="background:#f0f0f0;"|'''C1 well position''' | | align="center" style="background:#f0f0f0;"|'''C1 well position''' | ||
| align="center" style="background:#f0f0f0;"|'''3''' | | align="center" style="background:#f0f0f0;"|'''3''' |
Revision as of 18:10, 29 October 2015
PGP1-iPS derived cardiomyocyte RNA sequencing
- sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube
- prepared the C1 reaction according to manufacture protocol:
- Picogreen quantification
C1 well position | 3 | 2 | 1 | 49 | 50 | 51 | 6 | 5 | 4 | 52 | 53 | 54 |
concentration (ng/ul) | 0.730 | 0.743 | 0.631 | 0.883 | 0.685 | 0.714 | 0.751 | 1.773 | 1.818 | 0.875 | 0.950 | 0.876 |
C1 well position | 9 | 8 | 7 | 55 | 56 | 57 | 12 | 11 | 10 | 58 | 59 | 60 |
concentration (ng/ul) | 1.848 | 1.646 | 0.799 | 0.834 | 0.783 | 0.679 | 1.515 | 1.780 | 1.418 | 1.822 | 0.796 | 0.810 |
C1 well position | 15 | 14 | 13 | 61 | 62 | 63 | 18 | 17 | 16 | 64 | 65 | 66 |
concentration (ng/ul) | 0.858 | 2.430 | 0.000 | 0.806 | 0.743 | 0.810 | 0.775 | 1.944 | 0.733 | 2.094 | 0.958 | 0.818 |
C1 well position | 21 | 20 | 19 | 67 | 68 | 69 | 24 | 23 | 22 | 70 | 71 | 72 |
concentration (ng/ul) | 1.833 | 1.461 | 1.841 | 0.825 | 0.751 | 0.816 | 0.857 | 2.390 | 0.814 | 0.865 | 0.888 | 0.881 |
C1 well position | 25 | 26 | 27 | 75 | 74 | 73 | 28 | 29 | 30 | 78 | 77 | 76 |
concentration (ng/ul) | 2.377 | 1.582 | 2.141 | 1.906 | 0.892 | 0.708 | 0.000 | 0.000 | 1.614 | 0.881 | 0.795 | 0.919 |
C1 well position | 31 | 32 | 33 | 81 | 80 | 79 | 34 | 35 | 36 | 84 | 83 | 82 |
concentration (ng/ul) | 2.012 | 3.324 | 0.000 | 0.788 | 0.919 | 0.780 | 0.831 | 0.000 | 0.548 | 0.847 | 0.873 | 0.843 |
C1 well position | 37 | 38 | 39 | 87 | 86 | 85 | 40 | 41 | 42 | 90 | 89 | 88 |
concentration (ng/ul) | 2.617 | 1.585 | 2.158 | 0.810 | 0.793 | 0.710 | 0.000 | 2.048 | 0.073 | 0.853 | 0.749 | 0.692 |
C1 well position | 43 | 44 | 45 | 93 | 92 | 91 | 46 | 47 | 48 | 96 | 95 | 94 |
concentration (ng/ul) | 1.750 | 0.863 | 0.669 | 0.773 | 0.853 | 0.795 | 0.000 | 0.853 | 0.344 | 0.658 | 0.658 | 0.658 |
- Dilute C1 harvested samples to 0.2ng/ul according to above quantification results
Tagmentation (Nextera XT Tagment Amplicon tube)
- Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice
- Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex until all particulates are resuspended
- After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge
- Add 2.5 μl TD Buffer to each well to be used in this assay
- Add 1.25 μl diluted input DNA at 0.2 ng/μl (1 ng total) from above step
- Add 1.25 μl ATM to the wells containing input DNA and TD Buffer.
- Centrifuge at 280 × g at 20°C for 1 minute.
- Place in a thermal cycler and run the following program: 55°C for 5 minutes -> Hold at 10°C
- When the sample reaches 10°C, proceed immediately to Neutralize samples as the transposome is still active and add 1.25 μl NT Buffer
- vortex at medium speed briefly
- Centrifuge at 280 × g at 20°C for 1 minute.
- Leave at room temperature for 5 minutes.
PCR amplification of tagmentation treated DNA
- add 1.25 μl index 2 primers (white caps)
- add 1.25 μl index 1 primers (orange caps)
- add 3.75 ul NPM (amplification mix)
- Centrifuge at 280 × g at 20°C for 1 minute.
- Perform PCR using the following program on a thermal cycler:
- 72°C for 3 minutes
- 95°C for 30 seconds
- 12 cycles of: 95°C for 10 seconds, 55°C for 30 seconds, 72°C for 30 seconds
- 72°C for 5 minutes
- Hold at 10°C
- pooling cDNA libraries together by taking 4ul from each well
- Ampure bead XP purification with 0.8x concentration of bead, and elute in 50ul of H2O
- gel validation of bands from 250-800bp
- QPCR quantification