Blue:RNA-Seq Experiments:smarter test: Difference between revisions
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:::*72C 6min | :::*72C 6min | ||
::*72C 5min | ::*72C 5min | ||
== RNAseIII-Fragmented Library Preparation == | |||
:'''RNA PREPARATION''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|PAP RNA ||||||||||4.1ul | |||
|- | |||
|10mM dNTP Mix||||||||||1ul | |||
|- | |||
|1uM T20V.ID ||||||||||0.9ul | |||
|- | |||
|} | |||
:::''Incubate @ 65C 3 min'' | |||
:::''Transfer to Ice'' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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''' | |||
::#Add 25ul beads per well | |||
::#Allow to bind 15 min | |||
::#Remove supernatant and wash 2x with 1ml 80% EtOH | |||
::#Air Dry 10min or until cracking first occurs | |||
::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube | |||
:'''TSO (x2)''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|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)''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|TSO Reaction||||||||||11ul | |||
|- | |||
|2x Buffer||||||||||25ul | |||
|- | |||
|10uM P1-STRT (Smarter TSO)||||||||||1ul | |||
|- | |||
|10uM PCR_R_N2_id ||||||||||1ul | |||
|- | |||
|H2O||||||||||12ul | |||
|- | |||
|Total||||||||||50ul | |||
|- | |||
|} | |||
::{| {{table}} | |||
|- | |||
|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 == | == RNAseIII-Fragmented Library Preparation == |
Revision as of 00:49, 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 | Smarter TSO (In-house) | SMART CDS (In-house) | Smarter Full-length totoRNAseq | Read 1 and N2 barcode read | ||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented-PolyA | Smarter TSO (In-house) | SMART CDS (In-house) | Smarter Full-length totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented-PolyA | Smarter TSO (In-house) | SMART CDS (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 | Smarter TSO (In-house) | SMART CDS (In-house) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented | Smarter TSO (In-house) | SMART CDS (In-house) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented | Smarter TSO (In-house) | SMART CDS (In-house) | Clontech Smarter | |||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented | Smarter TSO (In-house) | SMART CDS (In-house) | Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented | Smarter TSO (In-house) | SMART CDS (In-house) | Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented | Smarter TSO (In-house) | SMART CDS (In-house) | Smarter totoRNAseq | |||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented | Smarter TSO (In-house) | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented | Smarter TSO (In-house) | T20V.id | N2_id | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented | Smarter TSO (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
Smarter Library Preparation (Unfragmented)
As per Clontech Kit using Kit or In-house reagents:
- RNA PREPARATION
Volume 2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul Dilution Buffer 2.375ul 3' SMART CDS Primer II A (12uM) 1.0ul RNase Inhibitor 0.125ul Total 4.5ul
- Incubate @ 72C 3 min
- Transfer to ice
Volume 5xRT Buffer 2ul 100mM DTT 0.25ul dNTP (10mM) 1ul Smarter II A Oligonucleotide (12uM) 1ul RNase Inhibitor (40U/ul) 0.25ul Smarter MMLV (100U/ul) 1.0ul (0.5 + 0.5 dH2O for in-house) Total 10ul
- Incubate @ 42C 90 min
- Incubate @ 70C 10 min
- Beads Purification
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- 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
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
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- 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
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- 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
- 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
- Add 25ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- 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
Results of Amplification
- Ampure Beads Purify (0.8x)
- Resuspend in 10ul
- Run 1ul on gel