Blue:RNA-Seq Experiments:07282014

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Revision as of 20:28, 1 August 2014 by >B1lake (→‎NexteraXT)
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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