Blue:RNA-Seq Experiments:12102013: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Library Conc'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
|-
|-
| BL_||UHRR/ERCC||10pg - RNase3 15s (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id89-90||totoRNAseq||||
| BL_totoRNAseq_10pgUHRR_Dec10||UHRR/ERCC||10pg - RNase3 15s (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id89-90||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10pg - RNase3 30s (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id91-92||totoRNAseq||||
| ||UHRR/ERCC||10pg - RNase3 30s (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id91-92||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id93-94||totoRNAseq||||
| ||UHRR/ERCC||10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id93-94||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10pg - RNase3 5min (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id95-96||totoRNAseq||||
| ||UHRR/ERCC||10pg - RNase3 5min (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id95-96||totoRNAseq||||
|-
|-
|}
|}
Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]


== Purpose ==
== Purpose ==
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[[File:12102013_PCR1_gel1.jpg]]
[[File:12102013_PCR1_gel1.jpg]]
Results:  Looks like the optimal fragmentation time is as low as 15s


== Final Library Preparation ==
== Final Library Preparation ==


*Beads purified twice at (0.8x), re-suspended in 10ul
*Beads purified twice at (0.8x), re-suspended in 10ul
*PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964. Compared with 1ng condition (Note: using 0.5ul of final 1ng combined library from 12062013):
[[File:12102013_PCR2_ERCC-3.jpg]]
[[File:12102013_PCR2_ERCC-5.jpg]]
[[File:12102013_PCR2_ERCC-30s.jpg]]
Results: Can see that the 3' is better represented than the 5' for 10pg.  In the 3' analysis it looks like 30s-1min are the better conditions, while in the 5' analysis the 15s-30s appears optimal.  Therefore, 30s may represent the universally best condition for 10pg RNA fragmentation.
*Combined id89-96 (2ul of each)
*Ran 1ul onto gel:
[[File:12102013_PCR1_gel2.jpg]]

Latest revision as of 01:29, 13 December 2013

RNA-Seq Library Information[edit]

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Type of Seq Reads Library Conc
BL_totoRNAseq_10pgUHRR_Dec10 UHRR/ERCC 10pg - RNase3 15s (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id89-90 totoRNAseq
UHRR/ERCC 10pg - RNase3 30s (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id91-92 totoRNAseq
UHRR/ERCC 10pg - RNase3 1min (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id93-94 totoRNAseq
UHRR/ERCC 10pg - RNase3 5min (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id95-96 totoRNAseq

Primer sequences [1]

Purpose[edit]

  • Test optimal RNaseIII fragmentation time for 10pg RNA based on 12062013
  • Use ERCCs to determine level of transcript amplification between libraries

RNAseIII-Fragmented Library Preparation[edit]

RNA
Volume
UHRR 0.02ng/ul 0.5ul
ERCC 1:10E6 0.2ul
0.1uM T20VN 0.1ul
dH2O 0.2ul
Total 1ul


Incubate @ 72C 3 min
Incubate @ 37C 1 min


Volume
10x RNaseIII buffer 0.2ul
RNase III 0.1ul
dH20 0.7ul
Total 2ul


Incubate @ 37C 15s-5min


3' Tailing
Volume
fRNA 2ul
5x RT buffer 0.5ul
1mM ATP 0.5ul
600U/ul Affymetrix PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.6ul
Total 4ul


Incubate @ 37C 10 min
Add 1ul of 0.1uM T20VN


Reverse Transcription
Volume
PAP RNA 5ul
5xRT Buffer 1.5ul
10mM dNTP Mix 1ul
100mM DTT 0.25ul
Betaine (5M) 2ul
Smarter MMLV (200U/ul) 0.25ul
Total 10ul


Incubate @ 42C 30 min


Beads Purification
  1. Add 15ul beads per well
  2. Allow to bind 15 min
  3. Remove supernatant and wash 2x with 1ml 80% EtOH
  4. Air Dry 10min or until cracking first occurs
  5. Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin to 3ul


TSO Reaction
Volume
Purified cDNA 3ul
5x RT buffer [MMLV] 2ul
10mM dNTP mix 1ul
100mM DTT 0.25ul
10uM TSO 1ul
Betaine (5M) 2ul
RNase Inhibitor 0.25ul
Smarter MMLV 0.5ul
Total 10ul


Incubate @42C 10min
Incubate @70C 10min


PCR (PhusionHF)
Volume
TSO Reaction 10ul
2x Buffer 25ul
10uM P1-STRT 1ul
10uM PCR_R_N2_id 1ul
H2O 13ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 15x (10pg)
72C, 5min

Results of Amplification[edit]

  • Run 5ul on gel:


File:12102013 PCR1 gel1.jpg


Results: Looks like the optimal fragmentation time is as low as 15s

Final Library Preparation[edit]

  • Beads purified twice at (0.8x), re-suspended in 10ul
  • PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964. Compared with 1ng condition (Note: using 0.5ul of final 1ng combined library from 12062013):

File:12102013 PCR2 ERCC-3.jpg

File:12102013 PCR2 ERCC-5.jpg

File:12102013 PCR2 ERCC-30s.jpg

Results: Can see that the 3' is better represented than the 5' for 10pg. In the 3' analysis it looks like 30s-1min are the better conditions, while in the 5' analysis the 15s-30s appears optimal. Therefore, 30s may represent the universally best condition for 10pg RNA fragmentation.


  • Combined id89-96 (2ul of each)
  • Ran 1ul onto gel:

File:12102013 PCR1 gel2.jpg