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

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>Zsakura2
(Created page with "=PGP1-iPS derived cardiomyocyte RNA sequencing= *sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube *prepared the")
 
>Zsakura2
Line 1: Line 1:
=PGP1-iPS derived cardiomyocyte RNA sequencing=
=PGP1-iPS derived cardiomyocyte RNA sequencing=
*sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube
*sorted ~15,000 Tmem+VCAM1+ (day 10-12) PGP1iPS differentiated cardiomyocyte into 1.5ml microcentrifuge tube
*prepared the
*prepared the C1 reaction according to manufacture protocol:
*Picogreen quantification
*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

Revision as of 18:06, 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
  • 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