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

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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''
Line 89: Line 89:
==Library Summary==
==Library Summary==


{| {{table}}
{| {{table}} border
| align="center" style="background:#f0f0f0;"|'''Group'''
| align="center" style="background:#f0f0f0;"|'''Group'''
| align="center" style="background:#f0f0f0;"|'''  |    '''
| align="center" style="background:#f0f0f0;"|'''Sample Name'''
| align="center" style="background:#f0f0f0;"|'''Sample Name'''
| align="center" style="background:#f0f0f0;"|'''  |    '''
| align="center" style="background:#f0f0f0;"|'''Nextera_Ind'''
| align="center" style="background:#f0f0f0;"|'''Nextera_Ind'''
|-
|-
| 1||1_PBMC_C03|| ||1
| 1||1_PBMC_C03||1
|-
|-
| 1||1_PBMC_C09|| ||2
| 1||1_PBMC_C09||2
|-
|-
| 1||1_PBMC_C15|| ||3
| 1||1_PBMC_C15||3
|-
|-
| 1||1_PBMC_C25|| ||4
| 1||1_PBMC_C25||4
|-
|-
| 1||1_PBMC_C43|| ||5
| 1||1_PBMC_C43||5
|-
|-
| 1||1_PBMC_C14|| ||6
| 1||1_PBMC_C14||6
|-
|-
| 1||1_PBMC_C20|| ||7
| 1||1_PBMC_C20||7
|-
|-
| 1||1_PBMC_C26|| ||8
| 1||1_PBMC_C26||8
|-
|-
| 1||1_PBMC_C01|| ||9
| 1||1_PBMC_C01||9
|-
|-
| 1||1_PBMC_C39|| ||10
| 1||1_PBMC_C39||10
|-
|-
| 1||1_PBMC_C61|| ||11
| 1||1_PBMC_C61||11
|-
|-
| 1||1_PBMC_C87|| ||12
| 1||1_PBMC_C87||12
|-
|-
| 1||1_PBMC_C56|| ||13
| 1||1_PBMC_C56||13
|-
|-
| 1||1_PBMC_C68|| ||14
| 1||1_PBMC_C68||14
|-
|-
| 1||1_PBMC_C74|| ||15
| 1||1_PBMC_C74||15
|-
|-
| 1||1_PBMC_C80|| ||16
| 1||1_PBMC_C80||16
|-
|-
| 1||1_PBMC_C92|| ||17
| 1||1_PBMC_C92||17
|-
|-
| 1||1_PBMC_C51|| ||18
| 1||1_PBMC_C51||18
|-
|-
| 1||1_PBMC_C57|| ||19
| 1||1_PBMC_C57||19
|-
|-
| 1||1_PBMC_C63|| ||20
| 1||1_PBMC_C63||20
|-
|-
| 2||1_PBMC_C69|| ||1
| 2||1_PBMC_C69||1
|-
|-
| 2||1_PBMC_C06|| ||2
| 2||1_PBMC_C06||2
|-
|-
| 2||1_PBMC_C18|| ||3
| 2||1_PBMC_C18||3
|-
|-
| 2||1_PBMC_C24|| ||4
| 2||1_PBMC_C24||4
|-
|-
| 2||1_PBMC_C28|| ||5
| 2||1_PBMC_C28||5
|-
|-
| 2||1_PBMC_C34|| ||6
| 2||1_PBMC_C34||6
|-
|-
| 2||1_PBMC_C46|| ||7
| 2||1_PBMC_C46||7
|-
|-
| 2||1_PBMC_C17|| ||8
| 2||1_PBMC_C17||8
|-
|-
| 2||1_PBMC_C35|| ||9
| 2||1_PBMC_C35|||9
|-
|-
| 2||1_PBMC_C41|| ||10
| 2||1_PBMC_C41||10
|-
|-
| 2||1_PBMC_C04|| ||11
| 2||1_PBMC_C04||11
|-
|-
| 2||1_PBMC_C48|| ||12
| 2||1_PBMC_C48||12
|-
|-
| 2||1_PBMC_C64|| ||13
| 2||1_PBMC_C64||13
|-
|-
| 2||1_PBMC_C78|| ||14
| 2||1_PBMC_C78||14
|-
|-
| 2||1_PBMC_C90|| ||15
| 2||1_PBMC_C90||15
|-
|-
| 2||1_PBMC_C53|| ||16
| 2||1_PBMC_C53||16
|-
|-
| 2||1_PBMC_C65|| ||17
| 2||1_PBMC_C65||17
|-
|-
| 2||1_PBMC_C54|| ||18
| 2||1_PBMC_C54||18
|-
|-
| 2||1_PBMC_C88|| ||19
| 2||1_PBMC_C88||19
|-
|-
| 2||1_PBMC_C94|| ||20
| 2||1_PBMC_C94||20
|-
|-
| TubeCont||CD4+|| ||21
| TubeCont||CD4+||21
|-
|-
| TubeCont||CD19+|| ||22
| TubeCont||CD19+||22
|-
|-
| TubeCont||PBMC|| ||23
| TubeCont||PBMC||23
|-
|-
|}
|}

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