Blue:RNA-Seq Experiments:07282014: Difference between revisions
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==SmartPlus on Mouse Nuclei== | ==SmartPlus on Mouse Spinal Nuclei (Chun Lab)== | ||
Obtained sorted nuclei from Aran: | Obtained sorted nuclei from Aran: | ||
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#5000 nuclei sorted into BSA buffer (2x) | #5000 nuclei sorted into BSA buffer (2x) | ||
*stored 100/500 sorts at -80C. | *stored 100/500 sorts at -80C. Removed 2 of each, vacuum spun down to 1ul for each. | ||
*RNA extraction of 5000 using Zymo Micro RNA Kit (including DNAse treatment). Eluted in 6ul and vacuum spun down to 2ul. | *RNA extraction of 5000 using Zymo Micro RNA Kit (including DNAse treatment). Eluted in 6ul and vacuum spun down to 2ul. | ||
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* | * Yields for the RNA 1 and 2 samples are too high (expect more of that seen for the 100-500 nuclei), therefore repeated PCR on RT reaction using 3 less cycles (18 total) | ||
Line 45: | Line 45: | ||
|- | |- | ||
| RNA 2||4.34 | | RNA 2||4.34 | ||
|} | |||
==NexteraXT== | |||
*cDNA diluted to 0.2ng/ul | |||
''Nextera Tagmentation'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Tagment DNA Buffer||||||||||2.5ul | |||
|- | |||
|Amplification Tagment Mix||||||||||1.25ul | |||
|- | |||
|Diluted Sample||||||||||1.25ul | |||
|- | |||
|Total||||||||||5ul | |||
|- | |||
|} | |||
::*Vortex and spin | |||
::*Incubate 55C 10 min | |||
::*Hold 10C | |||
::*Once sample reaches 10C add 1.25ul of NT buffer in block | |||
::*Vortex and spin | |||
''Nextera Amplification'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Tagmented Sample||||||||||6.25ul | |||
|- | |||
|Nextera PCR Mix||||||||||3.75ul | |||
|- | |||
|S501 Primer||||||||||1.25ul | |||
|- | |||
|N7id(1-24) Primer||||||||||1.25ul | |||
|- | |||
|Total||||||||||12.5ul | |||
|- | |||
|} | |||
::''Mix and centrifuge'' | |||
::{| {{table}} | |||
|- | |||
|72C 3 min|||||||||| | |||
|- | |||
|95C 30s|||||||||| | |||
|- | |||
|95C 10s -> 55C 30s -> 72C 60s||||||||||12x | |||
|- | |||
|72C, 5min | |||
|- | |||
|} | |||
*Beads purified (0.8x) twice | |||
*Resuspended in 10ul | |||
*Ran 1ul on gel: | |||
[[File:07282014_PCR1_Gel1.jpg|300px]] | |||
{| {{table}} border | |||
| align="center" style="background:#f0f0f0;"|'''N7xx''' | |||
| align="center" style="background:#f0f0f0;"|'''Sample''' | |||
| align="center" style="background:#f0f0f0;"|'''Barcode''' | |||
| align="center" style="background:#f0f0f0;"|'''S5xx''' | |||
|- | |||
| 1||4960 RNA Extracted (5000 nuc)||CGTGAT||1 | |||
|- | |||
| 2||5075 RNA Extracted (5000 nuc)||ACATCG||1 | |||
|- | |||
| 3||4960 - 100 Nuc||CGACTC||1 | |||
|- | |||
| 4||4960 - 100 Nuc||TGGTCA||1 | |||
|- | |||
| 5||4960 - 500 Nuc||CACTGT||1 | |||
|- | |||
| 6||4960 - 500 Nuc||ATTGGC||1 | |||
|- | |||
| | |||
|} | |} |
Latest revision as of 20:28, 1 August 2014
SmartPlus on Mouse Spinal Nuclei (Chun Lab)[edit]
Obtained sorted nuclei from Aran:
- 100 nuclei sorted directly into 2ul clontech dilution buffer (4x)
- 500 nuclei sorted directly into 2ul clontech dilution buffer (4x)
- 5000 nuclei sorted into BSA buffer (2x)
- stored 100/500 sorts at -80C. Removed 2 of each, vacuum spun down to 1ul for each.
- RNA extraction of 5000 using Zymo Micro RNA Kit (including DNAse treatment). Eluted in 6ul and vacuum spun down to 2ul.
- Performed SmartPlus protocol as per C1 tube control protocol on 1ul of above samples (only modification is addition of randomer) using 21 cycles for PCR
- cDNA Yields:
Sample | Sample Concentration (ng/ul) |
NC | 0.999 |
RNA 1 | 72.1 |
RNA 2 | 40.0 |
100 nuc 1 | 7.93 |
100 nuc 2 | 8.75 |
500 nuc 1 | 5.20 |
500 nuc 2 | 6.25 |
- Yields for the RNA 1 and 2 samples are too high (expect more of that seen for the 100-500 nuclei), therefore repeated PCR on RT reaction using 3 less cycles (18 total)
Sample | Sample Concentration (ng/ul) |
NC | 0.396 |
RNA 1 | 11.5 |
RNA 2 | 4.34 |
NexteraXT[edit]
- cDNA diluted to 0.2ng/ul
Nextera Tagmentation
Volume Tagment DNA Buffer 2.5ul Amplification Tagment Mix 1.25ul Diluted Sample 1.25ul Total 5ul
- Vortex and spin
- Incubate 55C 10 min
- Hold 10C
- Once sample reaches 10C add 1.25ul of NT buffer in block
- Vortex and spin
Nextera Amplification
Volume Tagmented Sample 6.25ul Nextera PCR Mix 3.75ul S501 Primer 1.25ul N7id(1-24) Primer 1.25ul Total 12.5ul
- Mix and centrifuge
72C 3 min 95C 30s 95C 10s -> 55C 30s -> 72C 60s 12x 72C, 5min
- Beads purified (0.8x) twice
- Resuspended in 10ul
- Ran 1ul on gel:
N7xx | Sample | Barcode | S5xx |
1 | 4960 RNA Extracted (5000 nuc) | CGTGAT | 1 |
2 | 5075 RNA Extracted (5000 nuc) | ACATCG | 1 |
3 | 4960 - 100 Nuc | CGACTC | 1 |
4 | 4960 - 100 Nuc | TGGTCA | 1 |
5 | 4960 - 500 Nuc | CACTGT | 1 |
6 | 4960 - 500 Nuc | ATTGGC | 1 |