Alice:Single/2015-10-23: Difference between revisions

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>Zsakura2
>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)

  1. Remove the ATM, TD, and input DNA from -25°C to -15°C storage and thaw on ice
  2. Visually inspect NT to make sure that there is no precipitate. If there is precipitate, vortex until all particulates are resuspended
  3. After thawing, mix reagents by gently inverting the tubes 3–5 times, followed by a brief spin in a microcentrifuge
  4. Add 2.5 μl TD Buffer to each well to be used in this assay
  5. Add 1.25 μl diluted input DNA at 0.2 ng/μl (1 ng total) from above step
  6. Add 1.25 μl ATM to the wells containing input DNA and TD Buffer.
  7. Centrifuge at 280 × g at 20°C for 1 minute.
  8. Place in a thermal cycler and run the following program: 55°C for 5 minutes -> Hold at 10°C
  9. When the sample reaches 10°C, proceed immediately to Neutralize samples as the transposome is still active and add 1.25 μl NT Buffer
  10. vortex at medium speed briefly
  11. Centrifuge at 280 × g at 20°C for 1 minute.
  12. Leave at room temperature for 5 minutes.

PCR amplification of tagmentation treated DNA

  1. add 1.25 μl index 2 primers (white caps)
  2. add 1.25 μl index 1 primers (orange caps)
  3. add 3.75 ul NPM (amplification mix)
  4. Centrifuge at 280 × g at 20°C for 1 minute.
  5. 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