Blue:RNA-Seq Experiments:09182013: Difference between revisions
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== RNA-Seq Library Information == | == RNA-Seq Library Information == | ||
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| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||1ng - Unfragmented | | BL_UHRR_||UHRR/ERCC||1ng - Unfragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter||Read 1 and N2 barcode read|| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||100pg - Unfragmented | | BL_UHRR_||UHRR/ERCC||100pg - Unfragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||10pg - Unfragmented | | BL_UHRR_||UHRR/ERCC||10pg - Unfragmented||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||1ng - Unfragmented|| | | BL_UHRR_||UHRR/ERCC||1ng - Unfragmented-PolyA||STRT_V2_3 (In-house)||STRT_V3_T30 (In-house)||||Smarter Full-length totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||100pg - Unfragmented|| | | 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 | | 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 - 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)||STRT_V3_T30 (In-house)||||Smarter totoRNAseq|||| | ||
Line 38: | Line 31: | ||
| BL_UHRR_||UHRR/ERCC||10pg - 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|| | | BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id||N2_id9||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id|| | | BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id||N2_id10||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id|| | | BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented||STRT_V2_3 (In-house)||T20V.id||N2_id11||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||1ng - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id|| | | BL_UHRR_||UHRR/ERCC||1ng - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id12||totoRNAseq|| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id|| | | BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id13||totoRNAseq|| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id|| | | BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id14||totoRNAseq|| | ||
|- | |- | ||
|} | |} | ||
Line 54: | Line 47: | ||
== Purpose == | == Purpose == | ||
*Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and | *Use variations of our protocol to evaluate the quality of library generation using Smart primers (kit or in-house) using ERCC QC assay | ||
*Examine efficiency of the modified TSO r03 ( | *Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and fragmentation using ERCC QC assay | ||
*Examine efficiency of the modified TSO r03 (resistant to Poly-A tailing) in a single step totoRNAseq reaction using ERCC QC assay | |||
== Library Preparation (Unfragmented) == | == Library Preparation (Unfragmented) == | ||
Line 70: | Line 64: | ||
|ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul | |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul | ||
|- | |- | ||
| | |STRT_V3_T30(10uM)||||||||||1.0ul | ||
|- | |- | ||
|dNTP (10mM)||||||||||1ul | |dNTP (10mM)||||||||||1ul | ||
Line 81: | Line 75: | ||
:::''Incubate @ | :::''Incubate @ 65C 3 min'' | ||
:::''Transfer to ice'' | :::''Transfer to ice'' | ||
Line 93: | Line 87: | ||
|100mM DTT||||||||||0.17ul | |100mM DTT||||||||||0.17ul | ||
|- | |- | ||
| | |STRT_V2_3 TSO (10uM)||||||||||1ul | ||
|- | |- | ||
|Betaine||||||||||2ul | |Betaine||||||||||2ul | ||
Line 128: | Line 122: | ||
|2x KAPA HiFi Hotstart ReadyMix||||||||||25ul | |2x KAPA HiFi Hotstart ReadyMix||||||||||25ul | ||
|- | |- | ||
| | |(10uM) STRT_PCR||||||||||1.0ul | ||
|- | |- | ||
|dH2O||||||||||14ul | |dH2O||||||||||14ul | ||
Line 164: | Line 158: | ||
|0.5U/ul PAP (1:10d in H2O)||||||||||0.25ul | |0.5U/ul PAP (1:10d in H2O)||||||||||0.25ul | ||
|- | |- | ||
|dH20||||||||||0. | |dH20||||||||||0.8ul | ||
|- | |- | ||
|Total||||||||||2.5ul | |Total||||||||||2.5ul | ||
Line 186: | Line 180: | ||
|10mM dNTP Mix||||||||||1ul | |10mM dNTP Mix||||||||||1ul | ||
|- | |- | ||
| | |10uM STRT_V3_T30||||||||||1ul | ||
|- | |- | ||
|dH2O||||||||||1.5ul | |dH2O||||||||||1.5ul | ||
Line 279: | Line 273: | ||
|2x KAPA HiFi Hotstart ReadyMix||||||||||25ul | |2x KAPA HiFi Hotstart ReadyMix||||||||||25ul | ||
|- | |- | ||
| | |STRT_PCR (10mM)||||||||||1.0ul | ||
|- | |- | ||
|dH2O||||||||||14ul | |dH2O||||||||||14ul | ||
Line 311: | Line 305: | ||
|10x RNaseIII Buffer||||||||||0.2ul | |10x RNaseIII Buffer||||||||||0.2ul | ||
|- | |- | ||
|1uM T20V. | |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30||||||||||0.1ul | ||
|- | |- | ||
|dH20||||||||||0.25ul | |dH20||||||||||0.25ul | ||
Line 354: | Line 348: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |fRNA||||||||||3.1ul | ||
|- | |- | ||
|5x RT Buffer||||||||||0.5ul | |5x RT Buffer||||||||||0.5ul | ||
Line 380: | Line 374: | ||
|10mM dNTP Mix||||||||||1ul | |10mM dNTP Mix||||||||||1ul | ||
|- | |- | ||
| | |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30||||||||||0.9ul | ||
|- | |- | ||
|} | |} | ||
Line 437: | Line 431: | ||
|100mM DTT||||||||||0.25ul | |100mM DTT||||||||||0.25ul | ||
|- | |- | ||
|10uM TSO||||||||||1ul | |10uM STRT_V2_3 TSO||||||||||1ul | ||
|- | |- | ||
|Betaine (5M)||||||||||2ul | |Betaine (5M)||||||||||2ul | ||
Line 461: | Line 455: | ||
:'''PCR ( | :'''PhusionHF PCR (for T20V.ID)''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 471: | Line 465: | ||
|2x Buffer||||||||||25ul | |2x Buffer||||||||||25ul | ||
|- | |- | ||
|10uM P1-STRT | |10uM P1-STRT||||||||||1ul | ||
|- | |- | ||
|10uM PCR_R_N2_id ||||||||||1ul | |10uM PCR_R_N2_id ||||||||||1ul | ||
Line 493: | Line 487: | ||
|- | |- | ||
|} | |} | ||
:'''KAPAHF PCR (STRT_V3_T30)''' | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|cDNA||||||||||11ul | |||
|- | |||
|2x KAPA HiFi Hotstart ReadyMix||||||||||25ul | |||
|- | |||
|STRT_PCR (10mM)||||||||||1.0ul | |||
|- | |||
|dH2O||||||||||13ul | |||
|- | |||
|Total||||||||||50ul | |||
|- | |||
|} | |||
::*98C 3min | |||
::*12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg): | |||
:::*98C 15 sec | |||
:::*67C 20sec | |||
:::*72C 30sec | |||
::*72C 5min | |||
== RNAseIII-Fragmented Library Preparation (1-Step) == | == RNAseIII-Fragmented Library Preparation (1-Step) == | ||
Line 503: | Line 525: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
|2ng/ul, | |2ng/ul, 200pg/ul, 20pg/ul UHRR||||||||||0.5ul | ||
|- | |- | ||
|ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul | |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul | ||
Line 509: | Line 531: | ||
|10x RNaseIII Buffer||||||||||0.15ul | |10x RNaseIII Buffer||||||||||0.15ul | ||
|- | |- | ||
|1uM T20V. | |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41||||||||||0.1ul | ||
|- | |- | ||
|Total||||||||||0.95ul | |Total||||||||||0.95ul | ||
Line 576: | Line 598: | ||
|10mM dNTP Mix||||||||||1ul | |10mM dNTP Mix||||||||||1ul | ||
|- | |- | ||
|1uM T20V. | |1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41||||||||||0.9ul | ||
|- | |- | ||
|} | |} | ||
Line 615: | Line 637: | ||
::''Add 1ul and incubate at 37C for 30 min'' | ::''Add 1ul and incubate at 37C for 30 min'' | ||
Line 653: | Line 674: | ||
== Results of Amplification == | == Results of Amplification == | ||
* | *Run 5ul on gel | ||
* | |||
* | |||
[[File:09182013_PCR1_gel1.jpg]] | |||
== ERCC Quality Control Analysis == | |||
*Comparison of Pre-amp and post-amp plots: | |||
**ERCC-130-5'F/R | |||
**ERCC-130-midF/R | |||
**ERCC-130-3'F/R | |||
*Controls (carried through RT and Beads purification but no TSO/PCR - made up to 50ul): | |||
**Unfragmented STRT | |||
**Unfragmented Smart | |||
**PolyA Unfragmented STRT | |||
**Fragmented STRT | |||
**Fragmented T20V | |||
*Amplified 1ul control and 1ul PCR (total volume 25ul) using Sybr-Green: | |||
::{| {{table}} | |||
|- | |||
|||||||||||<u>Volume</u> | |||
|- | |||
|Library||||||||||1ul | |||
|- | |||
|2xSybr Buffer||||||||||12.5ul | |||
|- | |||
|10uM F primer||||||||||0.5ul | |||
|- | |||
|10uM R Primer||||||||||0.5ul | |||
|- | |||
|dH2O||||||||||10.5ul | |||
|- | |||
|Total||||||||||25ul | |||
|- | |||
|} | |||
:::''95C 30sec'' | |||
:::''95C 10s -> 60C 20s -> 72C 30s'' => 40X | |||
:::''72C, 5min'' | |||
Loading Format for qPCR: | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Smart''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Smart-PolyA''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Smart-Frag''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
| align="center" style="background:#f0f0f0;"|'''Smart-T20''' | |||
| align="center" style="background:#f0f0f0;"|'''''' | |||
|- | |||
| ||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3'||align="center" style="background:#f0f0f0;"|5'||align="center" style="background:#f0f0f0;"|mid||align="center" style="background:#f0f0f0;"|3' | |||
|- | |||
| align="center" style="background:#f0f0f0;"|1ng PCR||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20||Smart-T20 | |||
|- | |||
| align="center" style="background:#f0f0f0;"|100pg PCR||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 | |||
|- | |||
| align="center" style="background:#f0f0f0;"|10pg PCR||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 | |||
|- | |||
| align="center" style="background:#f0f0f0;"|1ng RT||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 | |||
|- | |||
| align="center" style="background:#f0f0f0;"|100pg RT||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 | |||
|- | |||
| align="center" style="background:#f0f0f0;"|10pg RT||Smart||Smart||Smart||Smart-PolyA||Smart-PolyA||Smart-PolyA||Smart-Frag||Smart-Frag||Smart-Frag||Smart-Frag||Smart-T20||Smart-T20 | |||
| | |||
|- | |||
|} | |||
[[File:09182013_ERCC130_Smart.jpg]] | |||
Note: second chart compares 3' amplification for ERCC130 at different [RNA] | |||
[[File:09182013_ERCC130_Smart2.jpg]] | |||
[[File:09182013_ERCC130_Smart3.jpg]] | |||
== Final Library Preparation == | == Final Library Preparation == | ||
*Beads purified (0.8x) | |||
*Re-amplified whole libraries 6x (1ng) or 7x (100pg, 10pg) | |||
*Beads purified (0.8x) | |||
*Ran 1ul on gel: | |||
[[File:09182013_PCR2_gel1.jpg]] | |||
Notes: | |||
*Library amplifications seemed low - might be an issue with one of the reagents e.g. MMLV. Therefore, need to repeat library production | |||
*Combined Smart/r03 (id9/12) (1ng seen above) for sequencing |
Latest revision as of 00:39, 2 October 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_UHRR_ | UHRR/ERCC | 1ng - Unfragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter | Read 1 and N2 barcode read | ||
BL_UHRR_ | UHRR/ERCC | 100pg - Unfragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter | |||
BL_UHRR_ | UHRR/ERCC | 10pg - Unfragmented | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter | |||
BL_UHRR_ | UHRR/ERCC | 1ng - Unfragmented-PolyA | STRT_V2_3 (In-house) | STRT_V3_T30 (In-house) | Smarter Full-length totoRNAseq | |||
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 - 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_id9 | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented | STRT_V2_3 (In-house) | T20V.id | N2_id10 | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented | STRT_V2_3 (In-house) | T20V.id | N2_id11 | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 1ng - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id12 | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id13 | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg - RNaseIII Fragmented - 1 step | TSO_r03 | T20V.id | N2_id14 | totoRNAseq |
Purpose[edit]
- Use variations of our protocol to evaluate the quality of library generation using Smart primers (kit or in-house) using ERCC QC assay
- Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and fragmentation using ERCC QC assay
- Examine efficiency of the modified TSO r03 (resistant to Poly-A tailing) in a single step totoRNAseq reaction using ERCC QC assay
Library Preparation (Unfragmented)[edit]
- RNA PREPARATION
Volume 2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul STRT_V3_T30(10uM) 1.0ul dNTP (10mM) 1ul dH2O 1.8ul Total 4.5ul
- Incubate @ 65C 3 min
- Transfer to ice
Volume 5xRT Buffer 2ul 100mM DTT 0.17ul 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
- Add 15ul 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 (10uM) 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[edit]
- RNA PREPARATION
Volume 2ng/ul, 200pg/ul, 20pg/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 0.8ul Total 2.5ul
- Incubate @ 37C 5 min
- Transfer to Ice
- REVERSE TRANSCRIPTION
Volume PAP RNA 2.5ul 10mM dNTP Mix 1ul 10uM STRT_V3_T30 1ul dH2O 1.5ul
- Incubate @ 65C 3 min
- Transfer to Ice
Volume 5xRT Buffer 1.5ul 100mM DTT 0.17ul Betaine 2ul RNase Inhibitor (40U/ul) 0.1ul Smarter MMLV (200U/ul) 0.23ul Total 10ul
- Incubate @ 42C 90 min
- Beads Purification
- Add 15ul 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, vacuspin to 3ul
- TSO
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 Amplification
Volume cDNA 10ul 2x KAPA HiFi Hotstart ReadyMix 25ul STRT_PCR (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[edit]
- RNA PREPARATION
Volume 2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 10x RNaseIII Buffer 0.2ul 1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30 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 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(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30 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 15ul 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, vacuspin to 3ul
- TSO
Volume Purified cDNA 3ul 5x RT buffer [MMLV] 2ul 10mM dNTP mix 1ul 100mM DTT 0.25ul 10uM STRT_V2_3 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
- PhusionHF PCR (for T20V.ID)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM P1-STRT 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
- KAPAHF PCR (STRT_V3_T30)
Volume cDNA 11ul 2x KAPA HiFi Hotstart ReadyMix 25ul STRT_PCR (10mM) 1.0ul dH2O 13ul Total 50ul
- 98C 3min
- 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
- 98C 15 sec
- 67C 20sec
- 72C 30sec
- 72C 5min
RNAseIII-Fragmented Library Preparation (1-Step)[edit]
- RNA PREPARATION
Volume 2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul 10x RNaseIII Buffer 0.15ul 1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 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(1ng) or 0.1uM(100pg/10pg) T20V.id41 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[edit]
- Run 5ul on gel
ERCC Quality Control Analysis[edit]
- Comparison of Pre-amp and post-amp plots:
- ERCC-130-5'F/R
- ERCC-130-midF/R
- ERCC-130-3'F/R
- Controls (carried through RT and Beads purification but no TSO/PCR - made up to 50ul):
- Unfragmented STRT
- Unfragmented Smart
- PolyA Unfragmented STRT
- Fragmented STRT
- Fragmented T20V
- Amplified 1ul control and 1ul PCR (total volume 25ul) using Sybr-Green:
Volume Library 1ul 2xSybr Buffer 12.5ul 10uM F primer 0.5ul 10uM R Primer 0.5ul dH2O 10.5ul Total 25ul
- 95C 30sec
- 95C 10s -> 60C 20s -> 72C 30s => 40X
- 72C, 5min
Loading Format for qPCR:
' | ' | Smart | ' | ' | Smart-PolyA | ' | ' | Smart-Frag | ' | ' | Smart-T20 | ' | |
5' | mid | 3' | 5' | mid | 3' | 5' | mid | 3' | 5' | mid | 3' | ||
1ng PCR | Smart | Smart | Smart | Smart-PolyA | Smart-PolyA | Smart-PolyA | Smart-Frag | Smart-Frag | Smart-Frag | Smart-T20 | Smart-T20 | Smart-T20 | |
100pg PCR | Smart | Smart | Smart | Smart-PolyA | Smart-PolyA | Smart-PolyA | Smart-Frag | Smart-Frag | Smart-Frag | Smart-Frag | Smart-T20 | Smart-T20 | |
10pg PCR | Smart | Smart | Smart | Smart-PolyA | Smart-PolyA | Smart-PolyA | Smart-Frag | Smart-Frag | Smart-Frag | Smart-Frag | Smart-T20 | Smart-T20 | |
1ng RT | Smart | Smart | Smart | Smart-PolyA | Smart-PolyA | Smart-PolyA | Smart-Frag | Smart-Frag | Smart-Frag | Smart-Frag | Smart-T20 | Smart-T20 | |
100pg RT | Smart | Smart | Smart | Smart-PolyA | Smart-PolyA | Smart-PolyA | Smart-Frag | Smart-Frag | Smart-Frag | Smart-Frag | Smart-T20 | Smart-T20 | |
10pg RT | Smart | Smart | Smart | Smart-PolyA | Smart-PolyA | Smart-PolyA | Smart-Frag | Smart-Frag | Smart-Frag | Smart-Frag | Smart-T20 | Smart-T20 |
File:09182013 ERCC130 Smart.jpg
Note: second chart compares 3' amplification for ERCC130 at different [RNA]
File:09182013 ERCC130 Smart2.jpg
File:09182013 ERCC130 Smart3.jpg
Final Library Preparation[edit]
- Beads purified (0.8x)
- Re-amplified whole libraries 6x (1ng) or 7x (100pg, 10pg)
- Beads purified (0.8x)
- Ran 1ul on gel:
Notes:
- Library amplifications seemed low - might be an issue with one of the reagents e.g. MMLV. Therefore, need to repeat library production
- Combined Smart/r03 (id9/12) (1ng seen above) for sequencing