Blue:RNA-Seq Experiments:09182013: Difference between revisions

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>B1lake
>B1lake
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|3' CDS Primer IIA(12uM) or STRT_V3_T30(10uM)||||||||||1.0ul
|3' CDS Primer IIA(12uM) or STRT_V3_T30(10uM)||||||||||1.0ul
|-
|-
|RNase Inhibitor||||||||||0.125ul
|dNTP (10mM)||||||||||1ul
|-
|-
|dH2O||||||||||3.175ul
|dH2O||||||||||1.8ul
|-
|-
|Total||||||||||5ul
|Total||||||||||4.5ul
|-
|-
|}
|}
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|-
|-
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|RNA/3' primer||||||||||5ul
|-
|-
|5xRT Buffer||||||||||2ul  
|5xRT Buffer||||||||||2ul  
|-
|-
|100mM DTT||||||||||0.25ul
|100mM DTT||||||||||0.17ul
|-
|-
|dNTP (10mM)||||||||||1ul
|Smarter II A TSO(12uM) or STRT_V2_3 TSO (10uM)||||||||||1ul
|-
|-
|Smarter II A TSO(12uM) or STRT_V2_3 TSO||||||||||1ul
|Betaine||||||||||2ul
|-
|-
|RNase Inhibitor (40U/ul)||||||||||0.25ul
|RNase Inhibitor (40U/ul)||||||||||0.1ul
|-
|-
|Smarter MMLV (100U/ul)||||||||||0.5ul
|Smarter MMLV (100U/ul)||||||||||0.23ul
|-
|-
|Total||||||||||10ul
|Total||||||||||10ul

Revision as of 22:35, 17 September 2013

RNA-Seq Library Information

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Type of Seq Reads Library Conc
BL_UHRR_ UHRR/ERCC 1ng - Unfragmented-PolyA STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter Full-length totoRNAseq Read 1 and N2 barcode read
BL_UHRR_ UHRR/ERCC 100pg - Unfragmented-PolyA STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter Full-length totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - Unfragmented-PolyA STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter Full-length totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - Unfragmented Smarter TSO (Kit) SMART CDS (Kit) Clontech Smarter
BL_UHRR_ UHRR/ERCC 100pg - Unfragmented Smarter TSO (Kit) SMART CDS (Kit) Clontech Smarter
BL_UHRR_ UHRR/ERCC 10pg - Unfragmented Smarter TSO (Kit) SMART CDS (Kit) Clontech Smarter
BL_UHRR_ UHRR/ERCC 1ng - Unfragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Clontech Smarter
BL_UHRR_ UHRR/ERCC 100pg - Unfragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Clontech Smarter
BL_UHRR_ UHRR/ERCC 10pg - Unfragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Clontech Smarter
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg - RNaseIII Fragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - RNaseIII Fragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented STRT_V2_3 (In-house) T20V.id N2_id totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg - RNaseIII Fragmented STRT_V2_3 (In-house) T20V.id N2_id totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - RNaseIII Fragmented STRT_V2_3 (In-house) T20V.id N2_id totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RNaseIII Fragmented - 1 step TSO_r03 T20V.id N2_id totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg - RNaseIII Fragmented - 1 step TSO_r03 T20V.id N2_id totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - RNaseIII Fragmented - 1 step TSO_r03 T20V.id N2_id totoRNAseq

Purpose

  • Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and our fragmentation protocol using ERCC assay
  • Examine efficiency of the modified TSO r03 (unable to be Poly-A tailed) in a single step totoRNAseq reaction using ERCC assay

Library Preparation (Unfragmented)

RNA PREPARATION
Volume
2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
3' CDS Primer IIA(12uM) or STRT_V3_T30(10uM) 1.0ul
dNTP (10mM) 1ul
dH2O 1.8ul
Total 4.5ul


Incubate @ 72C 3 min
Transfer to ice


Volume
5xRT Buffer 2ul
100mM DTT 0.17ul
Smarter II A TSO(12uM) or STRT_V2_3 TSO (10uM) 1ul
Betaine 2ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (100U/ul) 0.23ul
Total 10ul


Incubate @ 42C 90 min
Incubate @ 70C 10 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 10ul dH2O, incubate 2min, transfer to new tube


PCR Amplification
Volume
cDNA 10ul
2x KAPA HiFi Hotstart ReadyMix 25ul
ISPCR (10mM) or STRT_PCR 1.0ul
dH2O 14ul
Total 50ul


  • 98C 3min
  • 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
  • 98C 15 sec
  • 67C 20sec
  • 72C 6min
  • 72C 5min

Poly-A Unfragmented Library Preparation

RNA PREPARATION
Volume
2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
5x RT Buffer 0.5ul
1mM ATP 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul
dH20 0.3ul
Total 2.5ul


Incubate @ 37C 5 min
Transfer to Ice


REVERSE TRANSCRIPTION
Volume
PAP RNA 2.5ul
10mM dNTP Mix 1ul
12uM CDS II A Primer 1ul
dH2O 2ul


Incubate @ 72C 3 min
Transfer to Ice


Volume
5xRT Buffer 1.5ul
100mM DTT 0.25ul
Smarter II A TSO (12uM) 1.0ul
RNase Inhibitor (40U/ul) 0.25ul
Smarter MMLV (200U/ul) 0.5ul
Total 10ul


Incubate @ 42C 90 min
Incubate @ 70C 10 min


Beads Purification
  1. Add 25ul 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 10ul dH2O, incubate 2min, transfer to new tube


PCR Amplification
Volume
cDNA 10ul
2x KAPA HiFi Hotstart ReadyMix 25ul
ISPCR primers (10mM) 1.0ul
dH2O 14ul
Total 50ul


  • 98C 3min
  • 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
  • 98C 15 sec
  • 67C 20sec
  • 72C 6min
  • 72C 5min

RNAseIII-Fragmented Library Preparation

RNA PREPARATION
Volume
2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
10x RNaseIII Buffer 0.2ul
1uM T20V.id1 0.1ul
dH20 0.25ul
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 1.25ul
RNase III 0.08ul
dH20 0.67ul
Total 2ul


Incubate @ 37C 10 min
Add 1.1ul dH2O
Incubate @ 65C 10 min


3' Tailing
Volume
PNK_fRNA 3.1ul
5x RT Buffer 0.5ul
1mM ATP 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul


Incubate @ 37C 5 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 4.1ul
10mM dNTP Mix 1ul
1uM T20V.ID 0.9ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 1.5ul
100mM DTT 0.17ul
Betaine (5M) 2ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (200U/ul) 0.23ul
Total 10ul


Incubate @ 42C 30 min


Beads Purification
  1. Add 25ul 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 (x2)
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


USER
Add 1ul and incubate at 37C for 30 min


PCR (PhusionHF)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM P1-STRT (Smarter TSO) 1ul
10uM PCR_R_N2_id 1ul
H2O 12ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 6x (1ng), 9x (100pg), 12x (10pg)
72C, 5min


RNAseIII-Fragmented Library Preparation (1-Step)

RNA PREPARATION
Volume
2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
10x RNaseIII Buffer 0.15ul
1uM T20V.id1 0.1ul
Total 0.95ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 0.95ul
RNase III 0.08ul
dH20 0.47ul
Total 1.5ul


Incubate @ 37C 10 min
Add 0.6ul dH2O
Incubate @ 65C 10 min


3' Tailing
Volume
fRNA 2.1ul
5x RT Buffer 0.5ul
1mM ATP 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul


Incubate @ 37C 5 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 3.1ul
10mM dNTP Mix 1ul
1uM T20V.ID 0.9ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 1.5ul
100mM DTT 0.17ul
Betaine (5M) 2ul
TSO.r03 (10uM) 1ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (200U/ul) 0.23ul
Total 10ul


Incubate @42C 30 min
Incubate @70C 10 min


USER
Add 1ul and incubate at 37C for 30 min


PCR (PhusionHF)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM PB_PCR_F 1ul
10uM PCR_R_N2_id 1ul
H2O 12ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 6x (1ng), 9x (100pg), 12x (10pg)
72C, 5min

Results of Amplification

  • Ampure Beads Purify (0.8x)
  • Resuspend in 10ul
  • Run 1ul on gel

Final Library Preparation