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 |
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=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)
- 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