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.  Vacuum spun down to 1ul for each.
*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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* Yield for the RNA is too high (expect more of that seen for the 100-500 nuclei), therefore repeated PCR using 3 less cycles (18 total)
* 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)




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|-
|-
| 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:

  1. 100 nuclei sorted directly into 2ul clontech dilution buffer (4x)
  2. 500 nuclei sorted directly into 2ul clontech dilution buffer (4x)
  3. 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:

File:07282014 PCR1 Gel1.jpg


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