Blue:RNA-Seq Experiments:11132013: Difference between revisions

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| align="center" style="background:#f0f0f0;"|'''Library Conc'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id1||totoRNAseq||||
| BL_totoRNAseq_10ngUHRR_Nov13||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id1||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/3'dATP (1:0.1)||TSO.r06||T20VN_PE_R||N2.id2||totoRNAseq||||
| ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/3'dATP (1:0.1)||TSO.r06||T20VN_PE_R||N2.id2||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/dATP (1:1)||TSO.r06||T20VN_PE_R||N2.id3||totoRNAseq||||
| ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/dATP (1:1)||TSO.r06||T20VN_PE_R||N2.id3||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id4||totoRNAseq||||
| ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP||TSO.r06||T20VN_PE_R||N2.id4||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/3'dATP (1:0.1)||TSO.r06||T20VN_PE_R||N2.id5||totoRNAseq||||
| ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/3'dATP (1:0.1)||TSO.r06||T20VN_PE_R||N2.id5||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/dATP (1:1)||TSO.r06||T20VN_PE_R||N2.id6||totoRNAseq||||
| ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/dATP (1:1)||TSO.r06||T20VN_PE_R||N2.id6||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP then add 3'dATP||TSO.r06||T20VN_PE_R||N2.id7||totoRNAseq||||
| ||UHRR/ERCC||10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP then add 3'dATP||TSO.r06||T20VN_PE_R||N2.id7||totoRNAseq||||
|-
|-
| BL_||UHRR/ERCC||10ng - RNase3 (Affy buffer)/Affymetrix PAP 10min ATP then add 3'dATP||TSO.r06||T20VN_PE_R||N2.id8||totoRNAseq||||
| ||UHRR/ERCC||10ng - RNase3 (Affy buffer)/Affymetrix PAP 10min ATP then add 3'dATP||TSO.r06||T20VN_PE_R||N2.id8||totoRNAseq||||
|-
|-
|}
|}
Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]


== Purpose ==
== Purpose ==


*Test original totoRNAseq protocol (using RNaseIII buffer and 5xRT for RnaseIII/PAP treatment) without heating to 65C to inactivate enzymes.  RNaseIII inactivation by RNase inhibitor addition and PAP limitation by 3'dATP or dATP addition
*Test original totoRNAseq protocol (using RNaseIII buffer for RnaseIII and addition of 5xRT buffer for PAP treatment) without heating to 65C to inactivate enzymes.  RNaseIII inactivation by RNase inhibitor addition and PAP limitation by 3'dATP or dATP addition
*Testing difference of Ambion PAP and Affymetrix PAP (note that Affymetrix PAP showed significant A tailing of TSO and was completely inhibited by 3'dATP)
*Testing difference of Ambion PAP and Affymetrix PAP (note that Affymetrix PAP showed significant A tailing of TSO and was completely inhibited by 3'dATP)
*Testing Affymetrix PAP in RnaseIII buffer/RT buffer versus Affymetrix buffer (5x stock: 100mM Tris-HCl, pH 7.0, 3.0mM MnCl2, 0.1mM EDTA, 1mM DTT, 500 μg/ml acetylated BSA, 50% glycerol) Note that the Affymetrix buffer does not have the high salts seen in other PAP buffers and may be more compatible with RNaseIII as well as RT


== RNAseIII-Fragmented Library Preparation ==
== RNAseIII-Fragmented Library Preparation ==
Line 216: Line 219:


[[File:11132013_PCR1_gel1.jpg]]
[[File:11132013_PCR1_gel1.jpg]]
'''ERCC Amplification'''
*Test the ERCC amplification by comparing PCR product with the RT reaction:
**PCR product (beads purified and in 10 ul: used 1:5 dilution)
**RT reaction (remaining half of RT: used 1:10 dilution)
**PCR:
***Sybr 12.5ul
***F primer 0.5ul
***R Primer 0.5ul
***Sample (id1-7) 0.5ul
***dH2O 10.5ul
'''PCR Plate format:'''
::{| {{table}}
|-
|Sample||<u>PCRF/R227</u>||<u>L127/R227</u>||<u>PCRF/R964</u>||<u>L857/R964</u>||<u>PCRF/R227</u>||<u>L127/R227</u>||<u>PCRF/R964</u>||<u>L857/R964</u>
|-
|Id1||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|Id2||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|Id3||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|Id4||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|Id5||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|Id6||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|Id7||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|NC||PCR||PCR||PCR||PCR||RT||RT||RT||RT
|-
|}
'''PCR1: ERCC 130 amplification using 5' primer set or 3' primer set:'''
[[File:11132013_PCR3_ERCC_1.jpg]]
'''PCR2: Amplification using ILMN_PCR_F primer and either the 5' or 3' reverse primer (Note that NC includes RT samples):'''
[[File:11132013_PCR3_ERCC_2.jpg]]
Results: While the Affymetrix PAP showed higher amplification in the original PCR reaction (P1-STRT/N2.id) and upon reamplification using ILMN_PCR_F/R, the Ambion PAP appears to amplify the ERCC more and showed no real bias between the 5' and 3' ends of the transcript.  However, the PCR curve looks more normal for the Affymetrix PAP.  Likely the Ambion results are questionable - the curves for the Affymetrix PAP look to be more accurate and reasonable.  I can conclude however that the Affymetrix libraries are real and represent amplified transcripts.


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


*Beads purified twice at (0.8x), re-suspend in 10ul
*Beads purified twice at (0.8x), re-suspend in 10ul
*Re-amplified 0.5ul using ILMN_PCR_F/R and SYBR:
[[File:11132013_PCR2_gel1.jpg]]
*Combined 2ul of original PCR of libraries 1-7.  Ran 1ul on gel:
[[File:11132013_PCR1_gel2.jpg]]

Latest revision as of 01:26, 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_10ngUHRR_Nov13 UHRR/ERCC 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP TSO.r06 T20VN_PE_R N2.id1 totoRNAseq
UHRR/ERCC 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/3'dATP (1:0.1) TSO.r06 T20VN_PE_R N2.id2 totoRNAseq
UHRR/ERCC 10ng - RNase3 (RIII buffer)/Ambion PAP 10min ATP/dATP (1:1) TSO.r06 T20VN_PE_R N2.id3 totoRNAseq
UHRR/ERCC 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP TSO.r06 T20VN_PE_R N2.id4 totoRNAseq
UHRR/ERCC 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/3'dATP (1:0.1) TSO.r06 T20VN_PE_R N2.id5 totoRNAseq
UHRR/ERCC 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP/dATP (1:1) TSO.r06 T20VN_PE_R N2.id6 totoRNAseq
UHRR/ERCC 10ng - RNase3 (RIII buffer)/Affymetrix PAP 10min ATP then add 3'dATP TSO.r06 T20VN_PE_R N2.id7 totoRNAseq
UHRR/ERCC 10ng - RNase3 (Affy buffer)/Affymetrix PAP 10min ATP then add 3'dATP TSO.r06 T20VN_PE_R N2.id8 totoRNAseq

Primer sequences [1]

Purpose[edit]

  • Test original totoRNAseq protocol (using RNaseIII buffer for RnaseIII and addition of 5xRT buffer for PAP treatment) without heating to 65C to inactivate enzymes. RNaseIII inactivation by RNase inhibitor addition and PAP limitation by 3'dATP or dATP addition
  • Testing difference of Ambion PAP and Affymetrix PAP (note that Affymetrix PAP showed significant A tailing of TSO and was completely inhibited by 3'dATP)
  • Testing Affymetrix PAP in RnaseIII buffer/RT buffer versus Affymetrix buffer (5x stock: 100mM Tris-HCl, pH 7.0, 3.0mM MnCl2, 0.1mM EDTA, 1mM DTT, 500 μg/ml acetylated BSA, 50% glycerol) Note that the Affymetrix buffer does not have the high salts seen in other PAP buffers and may be more compatible with RNaseIII as well as RT

RNAseIII-Fragmented Library Preparation[edit]

RNA
Volume
UHRR 20ng/ul 0.5ul
ERCC 1:10E3 0.2ul
1uM T20VN 0.1ul
dH2O 0.2ul
Total 1ul


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


Volume
10x RNaseIII buffer or 5x PAP buffer 0.2ul or 0.4ul
RNase III 0.1ul
dH20 0.7ul or 0.5ul
Total 2ul


Incubate @ 37C 5 min


3' Tailing
Volume
fRNA 2ul
5x RT buffer or 5x Affy PAP Buffer 0.5ul or 0.4ul
1mM ATP (or mix) 0.5ul
2U/ul Ambion PAP or 600U/ul Affymetrix PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.6ul or 0.7ul
Total 4ul


Incubate @ 37C 10 min
Add 1ul of 1uM T20VN or 0.5ul 2uM T20VN/0.05uM 3'dATP


Reverse Transcription
Volume
PAP RNA 5ul (or 4.5ul)
5xRT Buffer 1.5ul (or 2ul)
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


TSO on 1/2 RT 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
72C, 5min

Results of Amplification[edit]

  • Run 5ul on gel:


File:11132013 PCR1 gel1.jpg


ERCC Amplification

  • Test the ERCC amplification by comparing PCR product with the RT reaction:
    • PCR product (beads purified and in 10 ul: used 1:5 dilution)
    • RT reaction (remaining half of RT: used 1:10 dilution)
    • PCR:
      • Sybr 12.5ul
      • F primer 0.5ul
      • R Primer 0.5ul
      • Sample (id1-7) 0.5ul
      • dH2O 10.5ul


PCR Plate format:

Sample PCRF/R227 L127/R227 PCRF/R964 L857/R964 PCRF/R227 L127/R227 PCRF/R964 L857/R964
Id1 PCR PCR PCR PCR RT RT RT RT
Id2 PCR PCR PCR PCR RT RT RT RT
Id3 PCR PCR PCR PCR RT RT RT RT
Id4 PCR PCR PCR PCR RT RT RT RT
Id5 PCR PCR PCR PCR RT RT RT RT
Id6 PCR PCR PCR PCR RT RT RT RT
Id7 PCR PCR PCR PCR RT RT RT RT
NC PCR PCR PCR PCR RT RT RT RT


PCR1: ERCC 130 amplification using 5' primer set or 3' primer set:

File:11132013 PCR3 ERCC 1.jpg


PCR2: Amplification using ILMN_PCR_F primer and either the 5' or 3' reverse primer (Note that NC includes RT samples):

File:11132013 PCR3 ERCC 2.jpg


Results: While the Affymetrix PAP showed higher amplification in the original PCR reaction (P1-STRT/N2.id) and upon reamplification using ILMN_PCR_F/R, the Ambion PAP appears to amplify the ERCC more and showed no real bias between the 5' and 3' ends of the transcript. However, the PCR curve looks more normal for the Affymetrix PAP. Likely the Ambion results are questionable - the curves for the Affymetrix PAP look to be more accurate and reasonable. I can conclude however that the Affymetrix libraries are real and represent amplified transcripts.

Final Library Preparation[edit]

  • Beads purified twice at (0.8x), re-suspend in 10ul


  • Re-amplified 0.5ul using ILMN_PCR_F/R and SYBR:

File:11132013 PCR2 gel1.jpg


  • Combined 2ul of original PCR of libraries 1-7. Ran 1ul on gel:

File:11132013 PCR1 gel2.jpg