Blue:RNA-Seq Experiments:01082014: Difference between revisions

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>B1lake
(Created page with "== RNA-Seq Library Information == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Samples''' | ali...")
 
>B1lake
 
(13 intermediate revisions by the same user not shown)
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| align="center" style="background:#f0f0f0;"|'''Library Conc'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
|-
|-
| BL_totoRNAseq_10pgUHRR_Jan08||UHRR/ERCC||10pg - RNase3 15s (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id89-90||totoRNAseq||||
| BL_totoRNAseq_10pgUHRR_Jan08||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 1min||TSO.r06||T20VN_PE_R||N2.id1-2||totoRNAseq||||
|-
| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 2min||TSO.r06||T20VN_PE_R||N2.id3-4||totoRNAseq||||
|-
| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 4min||TSO.r06||T20VN_PE_R||N2.id5-6||totoRNAseq||||
|-
| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 6min||TSO.r06||T20VN_PE_R||N2.id7-8||totoRNAseq||||
|-
| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 8min||TSO.r06||T20VN_PE_R||N2.id9-10||totoRNAseq||||
|-
| ||UHRR/ERCC||10pg - RNase3 15s/Affymetrix PAP 10min||TSO.r06||T20VN_PE_R||N2.id11-12||totoRNAseq||||
|-
| ||UHRR/ERCC||1ng - RNase3 1min/Affymetrix PAP 10min||TSO.r06||T20VN_PE_R||N2.id13-14||totoRNAseq||||
|-
|-
|}
|}


Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]
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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|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|UHRR 0.02ng/ul ||||||||||0.5ul
|UHRR 2ng/ul or 0.02ng/ul ||||||||||0.5ul
|-
|-
|ERCC 1:10E6||||||||||0.2ul
|ERCC 1:10E4 or 1:10E6||||||||||0.2ul
|-
|-
|0.1uM T20VN||||||||||0.1ul
|0.1uM T20VN||||||||||0.1ul
Line 67: Line 79:




:::''Incubate @ 37C 15s''
:::''Incubate @ 37C 15s-1min''




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:::''Incubate @ 37C 10 min''
:::''Incubate @ 37C 1-10 min''


:::''Add 1ul of 0.1uM T20VN''
:::''Add 1ul of 0.1uM T20VN''
Line 129: Line 141:


::#Add 15ul beads per well
::#Add 15ul beads per well
::#Allow to bind 15 min, combine
::#Allow to bind 15 min  
::#Remove supernatant and wash 2x with 1ml 80% EtOH
::#Remove supernatant and wash 2x with 1ml 80% EtOH
::#Air Dry 10min or until cracking first occurs
::#Air Dry 10min or until cracking first occurs
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|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|-
|98C 10s -> 72C 20s||||||||||15x (10pg)
|98C 10s -> 72C 20s||||||||||9x (1ng); 15x (10pg)
|-
|-
|72C, 5min
|72C, 5min
|-
|-
|}
|}


== Results of Amplification ==
== Results of Amplification ==
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*Run 5ul on gel:
*Run 5ul on gel:


[[File:01082013_PCR1_Gel1.jpg|400px]]






 
Results:  Global amplification levels seem a little low, but otherwise looks fine.  Some variability with the 10pg that could be due to the beads purification stage following RT...
Results:   
 
 


== Final Library Preparation ==
== Final Library Preparation ==
Line 220: Line 228:
*PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964.  
*PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964.  


[[File:01082014_ERCC130_R227.jpg|400px]][[File:01082014_ERCC130_R964b.jpg|400px]]


Results:
Results:
*Still variable amplification levels seen with 10pg.  More consistency might be achieved by combining 10pg samples for beads purification prior to TSO/PCR
*Can see at least one of the 2min or 4 min showing similar levels as the 1ng (especially for the R964 - i.e.3'end) while longer PAP times tended to show poorer amplification curves... therefore, 4 min PAP may be sufficient for 10pg RNA quantities
*Its difficult to fully conclude optimal conditions due to the variability... hopefully this is an artifact of the single 10pg beads purification rather than inconsistency/inefficiency of the totoRNAseq reactions at these minimal RNA concentrations

Latest revision as of 17:51, 16 January 2014

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_Jan08 UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 1min TSO.r06 T20VN_PE_R N2.id1-2 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 2min TSO.r06 T20VN_PE_R N2.id3-4 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 4min TSO.r06 T20VN_PE_R N2.id5-6 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 6min TSO.r06 T20VN_PE_R N2.id7-8 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 8min TSO.r06 T20VN_PE_R N2.id9-10 totoRNAseq
UHRR/ERCC 10pg - RNase3 15s/Affymetrix PAP 10min TSO.r06 T20VN_PE_R N2.id11-12 totoRNAseq
UHRR/ERCC 1ng - RNase3 1min/Affymetrix PAP 10min TSO.r06 T20VN_PE_R N2.id13-14 totoRNAseq


Primer sequences [1]

Purpose[edit]

  • Test optimal PAP treatment 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 2ng/ul or 0.02ng/ul 0.5ul
ERCC 1:10E4 or 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-1min


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 1-10 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 9x (1ng); 15x (10pg)
72C, 5min

Results of Amplification[edit]

  • Run 5ul on gel:

File:01082013 PCR1 Gel1.jpg


Results: Global amplification levels seem a little low, but otherwise looks fine. Some variability with the 10pg that could be due to the beads purification stage following RT...

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.

File:01082014 ERCC130 R227.jpgFile:01082014 ERCC130 R964b.jpg

Results:

  • Still variable amplification levels seen with 10pg. More consistency might be achieved by combining 10pg samples for beads purification prior to TSO/PCR
  • Can see at least one of the 2min or 4 min showing similar levels as the 1ng (especially for the R964 - i.e.3'end) while longer PAP times tended to show poorer amplification curves... therefore, 4 min PAP may be sufficient for 10pg RNA quantities
  • Its difficult to fully conclude optimal conditions due to the variability... hopefully this is an artifact of the single 10pg beads purification rather than inconsistency/inefficiency of the totoRNAseq reactions at these minimal RNA concentrations