Blue:RNA-Seq Experiments:07022014: Difference between revisions
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| align="center" style="background:#f0f0f0;"|'''Group''' | | align="center" style="background:#f0f0f0;"|'''Group''' | ||
| align="center" style="background:#f0f0f0;"|'''Sample Name''' | | align="center" style="background:#f0f0f0;"|'''Sample Name''' | ||
| align="center" style="background:#f0f0f0;"|''' ''' | | align="center" style="background:#f0f0f0;"|''' ''' | ||
| align="center" style="background:#f0f0f0;"|'''Nextera_Ind''' | | align="center" style="background:#f0f0f0;"|'''Nextera_Ind''' | ||
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Revision as of 22:43, 10 July 2014
PBMC C1 Experiment
Extracted RNA from bulk samples using Zymo microRNA Kit and eluted in 6ul. Determined [RNA] using Qubit:
- CD4+ 26.1 ng/uL
- CD19+ 6.27 ng/uL
- 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:
- CD4+ 6.56ng/ul - dilute 1:22 for 0.3ng/ul
- CD19+ 7.60ng/ul - dilute 1:25 for 0.3ng/ul
- PBMC 5.02ng/ul - dilute 1:17 for 0.3ng/ul
- 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:
Library Summary
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 |