Blue:RNA-Seq Experiments:07022014: Difference between revisions

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*Combined samples 1-20 (group 1 with N7 ids 1-20)
*Combined samples 21-40 (group 2 with N7 ids 1-20)
*Combined tube controls
*Beads purified (0.8x) twice
*Resuspended Group 1 and Group 2 in 50ul; resuspended tube controls in 10ul
*Ran 1ul on gel:

Revision as of 21:52, 10 July 2014

PBMC C1 Experiment

Extracted RNA from bulk samples using Zymo microRNA Kit and eluted in 6ul. Determined [RNA] using Qubit:

  1. CD4+ 26.1 ng/uL
  2. CD19+ 6.27 ng/uL
  3. PBMC 95.8 ng/uL


Diluted to 1ng/ul and used 1ng as tube control for C1 run 20140702 (followed standard Smart+randomer C1 tube control protocol with 3 fewer cycles in last stage of PCR)

Qubit quantification of PCR product:

  1. CD4+ 6.56ng/ul - dilute 1:22 for 0.3ng/ul
  2. CD19+ 7.60ng/ul - dilute 1:25 for 0.3ng/ul
  3. PBMC 5.02ng/ul - dilute 1:17 for 0.3ng/ul
  4. Neg Cont 1.64ng/ul

NexteraXT

  • Single nuclei cDNA transfered to new 96-well plate using EpiMotion
  • cDNA for single nuclei used undiluted for Nextera:

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 10ul


Mix and centrifuge


72C 3 min
95C 30s
95C 10s -> 55C 30s -> 72C 60s 12x
72C, 5min


  • Combined samples 1-20 (group 1 with N7 ids 1-20)
  • Combined samples 21-40 (group 2 with N7 ids 1-20)
  • Combined tube controls
  • Beads purified (0.8x) twice
  • Resuspended Group 1 and Group 2 in 50ul; resuspended tube controls in 10ul
  • Ran 1ul on gel: