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

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>B1lake
>B1lake
 
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==NexteraXT==
==NexteraXT==


*Single nuclei cDNA transfered to new 96-well plate using EpiMotion
*Single nuclei cDNA (generated on C1 by Rui 05242014) transferred to new 96-well plate using EpiMotion
*cDNA for single nuclei used undiluted for Nextera:
*cDNA for single nuclei was used undiluted for NexteraXT:


''Nextera Tagmentation''
''Nextera Tagmentation''

Latest revision as of 01:03, 11 July 2014

PBMC C1 Experiment[edit]

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[edit]

  • Single nuclei cDNA (generated on C1 by Rui 05242014) transferred to new 96-well plate using EpiMotion
  • cDNA for single nuclei was used undiluted for NexteraXT:

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:

File:07022014 PCR1 Gel1.jpg

Library Summary[edit]

Group Sample Name Nextera_Ind
1 1_PBMC_C03 1
1 1_PBMC_C09 2
1 1_PBMC_C15 3
1 1_PBMC_C25 4
1 1_PBMC_C43 5
1 1_PBMC_C14 6
1 1_PBMC_C20 7
1 1_PBMC_C26 8
1 1_PBMC_C01 9
1 1_PBMC_C39 10
1 1_PBMC_C61 11
1 1_PBMC_C87 12
1 1_PBMC_C56 13
1 1_PBMC_C68 14
1 1_PBMC_C74 15
1 1_PBMC_C80 16
1 1_PBMC_C92 17
1 1_PBMC_C51 18
1 1_PBMC_C57 19
1 1_PBMC_C63 20
2 1_PBMC_C69 1
2 1_PBMC_C06 2
2 1_PBMC_C18 3
2 1_PBMC_C24 4
2 1_PBMC_C28 5
2 1_PBMC_C34 6
2 1_PBMC_C46 7
2 1_PBMC_C17 8
2 1_PBMC_C35 9
2 1_PBMC_C41 10
2 1_PBMC_C04 11
2 1_PBMC_C48 12
2 1_PBMC_C64 13
2 1_PBMC_C78 14
2 1_PBMC_C90 15
2 1_PBMC_C53 16
2 1_PBMC_C65 17
2 1_PBMC_C54 18
2 1_PBMC_C88 19
2 1_PBMC_C94 20
TubeCont CD4+ 21
TubeCont CD19+ 22
TubeCont PBMC 23