Blue:RNA-Seq Experiments:smarter test: Difference between revisions
Jump to navigation
Jump to search
>B1lake |
>B1lake |
||
(43 intermediate revisions by the same user not shown) | |||
Line 9: | Line 9: | ||
| align="center" style="background:#f0f0f0;"|'''TSO Primer''' | | align="center" style="background:#f0f0f0;"|'''TSO Primer''' | ||
| align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | | align="center" style="background:#f0f0f0;"|'''Bar Code Primers''' | ||
| align="center" style="background:#f0f0f0;"|''' | | align="center" style="background:#f0f0f0;"|'''PCR Primers''' | ||
| align="center" style="background:#f0f0f0;"|'''Number of Cycles Amp''' | |||
| align="center" style="background:#f0f0f0;"|'''Type of Seq''' | | align="center" style="background:#f0f0f0;"|'''Type of Seq''' | ||
| align="center" style="background:#f0f0f0;"|'''Reads''' | | align="center" style="background:#f0f0f0;"|'''Reads''' | ||
| align="center" style="background:#f0f0f0;"|'''Library Conc''' | | align="center" style="background:#f0f0f0;"|'''Library Conc''' | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||1ng - | | BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id1||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC|| | | BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id2||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC|| | | BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id3-4||18 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC|| | | BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id5-6||18 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC|| | | BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id7-8||21 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||10pg - | | BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id9-10||21 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| | | BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id11||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| | | BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id12||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| | | BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id13||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| | | BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id14||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC|| | | BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id15||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC|| | | BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id16||15 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC|| | | BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id17-18||18 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented | | BL_UHRR_||UHRR/ERCC||100pg (x2) - RNaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id19-20||18 Cycles||totoRNAseq|||| | ||
|- | |- | ||
| BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented - | | BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id21-22||21 Cycles||totoRNAseq|||| | ||
|- | |||
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id23-24||21 Cycles||totoRNAseq|||| | |||
|- | |||
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id25||15 Cycles||totoRNAseq|||| | |||
|- | |||
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id26||15 Cycles||totoRNAseq|||| | |||
|- | |||
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id27||15 Cycles||totoRNAseq|||| | |||
|- | |||
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id28||15 Cycles||totoRNAseq|||| | |||
|- | |- | ||
|} | |} | ||
[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List Primer List] | |||
UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = Human Pyramidal Nuclei sorted 09-23-13 | |||
== Purpose == | == Purpose == | ||
* | *Generate totoRNAseq libraries using several TSO to compare efficiencies | ||
* | *Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal nuclei | ||
== Library Preparation | == RNAseIII-Fragmented Library Preparation == | ||
:'''RNA | :'''RNA PREPARATION''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 60: | Line 76: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |2ng/ul, 200ng/ul, 20ng/ul UHRR||||||||||0.5ul | ||
|- | |- | ||
|ERCC 1:10E4||||||||||0.2ul | |ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul | ||
|- | |- | ||
|10x RNaseIII Buffer||||||||||0.2ul | |10x RNaseIII Buffer||||||||||0.2ul | ||
|- | |- | ||
|1uM T20V.id1 | |1uM or 0.1uM (10-100pg) T20V.id1 ||||||||||0.1ul | ||
|- | |- | ||
|dH20||||||||||0.25ul | |dH20||||||||||0.25ul | ||
Line 76: | Line 92: | ||
:::''Incubate @ 72C 3 min'' | :::''Incubate @ 72C 3 min'' | ||
:::''Incubate @ 37C 1min'' | :::''Incubate @ 37C 1min'' | ||
Line 96: | Line 111: | ||
:::''Incubate @ 37C 10 min'' | :::''Incubate @ 37C 5-10 min'' | ||
:::''Add 1.1ul dH2O | :::''Add 1.1ul dH2O | ||
Line 110: | Line 125: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |fRNA||||||||||3.1ul | ||
|- | |- | ||
|5x RT Buffer||||||||||0.5ul | |5x RT Buffer||||||||||0.5ul | ||
Line 124: | Line 139: | ||
:::''Transfer to Ice'' | :::''Transfer to Ice'' | ||
:'''Reverse Transcription''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 155: | Line 147: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
|PAP RNA ||||||||||4.1ul | |||
|PAP RNA | |||
|- | |- | ||
|10mM dNTP Mix||||||||||1ul | |10mM dNTP Mix||||||||||1ul | ||
|- | |- | ||
|1uM T20V.ID | |1uM or 0.1uM (10-100pg) T20V.ID ||||||||||0.9ul | ||
|- | |- | ||
|} | |} | ||
Line 216: | Line 166: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
|5xRT Buffer||||||||||1.5ul | |5xRT Buffer||||||||||1.5ul | ||
|- | |- | ||
|100mM DTT||||||||||0.17ul | |100mM DTT||||||||||0.17ul | ||
Line 240: | Line 190: | ||
::#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 | ||
::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube | ::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube - vacuspin 5 min to bring to 3ul | ||
:'''TSO | :'''TSO''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 293: | Line 242: | ||
|2x Buffer||||||||||25ul | |2x Buffer||||||||||25ul | ||
|- | |- | ||
|10uM | |10uM P1-STRT (Smarter TSO) or PB_PCR_F||||||||||1ul | ||
|- | |- | ||
|10uM | |10uM PCR_R_N2_id||||||||||1ul | ||
|- | |- | ||
|H2O||||||||||12ul | |H2O||||||||||12ul | ||
Line 310: | Line 259: | ||
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x | |98C 15s -> 58C 20s -> 72C 30s||||||||||6x | ||
|- | |- | ||
|98C 10s -> 72C 20s|||||||||| | |98C 10s -> 72C 20s||||||||||9x (1ng), 12x (100pg), 18x (10pg) | ||
|- | |- | ||
|72C, 5min | |72C, 5min | ||
Line 318: | Line 267: | ||
== Results of Amplification == | == Results of Amplification == | ||
* | *Ampure Beads Purify (0.8x) | ||
*Resuspend in 10ul | |||
*Run 1ul on gel | |||
* | |||
* | |||
== Final Library Preparation == | == Final Library Preparation == | ||
Latest revision as of 21:43, 26 September 2013
RNA-Seq Library Information[edit]
Library ID | Samples | Input RNA | TSO Primer | Bar Code Primers | PCR Primers | Number of Cycles Amp | Type of Seq | Reads | Library Conc |
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented 10 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id1 | 15 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented 5 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id2 | 15 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg (x2) - RNaseIII Fragmented 10 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id3-4 | 18 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg (x2) - RNaseIII Fragmented 5 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id5-6 | 18 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg (x2) - RNaseIII Fragmented 10 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id7-8 | 21 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg (x2) - RNaseIII Fragmented 5 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id9-10 | 21 Cycles | totoRNAseq | ||
BL_HuNu_ | HuNu/ERCC | 1ng - RnaseIII Fragmented 10 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id11 | 15 Cycles | totoRNAseq | ||
BL_HuNu_ | HuNu/ERCC | 1ng - RnaseIII Fragmented 5 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id12 | 15 Cycles | totoRNAseq | ||
BL_HuLCM_ | HuLCM/ERCC | 1ng - RnaseIII Fragmented 10 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id13 | 15 Cycles | totoRNAseq | ||
BL_HuLCM_ | HuLCM/ERCC | 1ng - RnaseIII Fragmented 5 min | TSO_r04_N6 | T20V.id1 | PB_PCR_F/N2_id14 | 15 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented 10 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id15 | 15 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 1ng - RnaseIII Fragmented 5 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id16 | 15 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg (x2) - RNaseIII Fragmented 10 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id17-18 | 18 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 100pg (x2) - RNaseIII Fragmented 5 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id19-20 | 18 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg (x2) - RNaseIII Fragmented 10 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id21-22 | 21 Cycles | totoRNAseq | ||
BL_UHRR_ | UHRR/ERCC | 10pg (x2) - RNaseIII Fragmented 5 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id23-24 | 21 Cycles | totoRNAseq | ||
BL_HuNu_ | HuNu/ERCC | 1ng - RnaseIII Fragmented 10 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id25 | 15 Cycles | totoRNAseq | ||
BL_HuNu_ | HuNu/ERCC | 1ng - RnaseIII Fragmented 5 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id26 | 15 Cycles | totoRNAseq | ||
BL_HuLCM_ | HuLCM/ERCC | 1ng - RnaseIII Fragmented 10 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id27 | 15 Cycles | totoRNAseq | ||
BL_HuLCM_ | HuLCM/ERCC | 1ng - RnaseIII Fragmented 5 min | TSO.r06 | T20V.id1 | P1-STRT/N2_id28 | 15 Cycles | totoRNAseq |
UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = Human Pyramidal Nuclei sorted 09-23-13
Purpose[edit]
- Generate totoRNAseq libraries using several TSO to compare efficiencies
- Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal nuclei
RNAseIII-Fragmented Library Preparation[edit]
- 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 or 0.1uM (10-100pg) 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 5-10 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 or 0.1uM (10-100pg) 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 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 5 min to bring 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 (PhusionHF)
Volume TSO Reaction 11ul 2x Buffer 25ul 10uM P1-STRT (Smarter TSO) or 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 9x (1ng), 12x (100pg), 18x (10pg) 72C, 5min
Results of Amplification[edit]
- Ampure Beads Purify (0.8x)
- Resuspend in 10ul
- Run 1ul on gel