Blue:RNA-Seq Experiments:smarter test: Difference between revisions

From ZhangLabWiki
Jump to navigation Jump to search
>B1lake
>B1lake
 
(18 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;"|'''Bar Code Primer 2'''
| 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 - Unfragmented-PolyA||Smarter TSO (In-house)||SMART CDS (In-house)||||Smarter Full-length totoRNAseq||Read 1 and N2 barcode read||
| 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||100pg - Unfragmented-PolyA||Smarter TSO (In-house)||SMART CDS (In-house)|||||Smarter Full-length 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||10pg - Unfragmented-PolyA||Smarter TSO (In-house)||SMART CDS (In-house)||||Smarter Full-length 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||1ng - Unfragmented||Smarter TSO (Kit)||SMART CDS (Kit)||||Clontech Smarter||||
| 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||100pg - Unfragmented||Smarter TSO (Kit)||SMART CDS (Kit)||||Clontech Smarter||||
| 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 - Unfragmented||Smarter TSO (Kit)||SMART CDS (Kit)||||Clontech Smarter||||
| 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_UHRR_||UHRR/ERCC||1ng - Unfragmented||Smarter TSO (In-house)||SMART CDS (In-house)||||Clontech Smarter||||
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id11||15 Cycles||totoRNAseq||||
|-
|-
| BL_UHRR_||UHRR/ERCC||100pg - Unfragmented||Smarter TSO (In-house)||SMART CDS (In-house)||||Clontech Smarter||||
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 5 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id12||15 Cycles||totoRNAseq||||
|-
|-
| BL_UHRR_||UHRR/ERCC||10pg - Unfragmented||Smarter TSO (In-house)||SMART CDS (In-house)||||Clontech Smarter||||
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 10 min||TSO_r04_N6||T20V.id1||PB_PCR_F/N2_id13||15 Cycles||totoRNAseq||||
|-
|-
| BL_UHRR_||UHRR/ERCC||1ng - RnaseIII Fragmented||Smarter TSO (In-house)||SMART CDS (In-house)||||Smarter 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||100pg - RNaseIII Fragmented||Smarter TSO (In-house)||SMART CDS (In-house)||||Smarter 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||10pg - RNaseIII Fragmented||Smarter TSO (In-house)||SMART CDS (In-house)||||Smarter 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||1ng - RnaseIII Fragmented||Smarter TSO (In-house)||T20V.id||N2_id||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 - RNaseIII Fragmented||Smarter TSO (In-house)||T20V.id||N2_id||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 - RNaseIII Fragmented||Smarter TSO (In-house)||T20V.id||N2_id||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||1ng - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id||totoRNAseq||
| BL_UHRR_||UHRR/ERCC||10pg (x2) - RNaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id23-24||21 Cycles||totoRNAseq||||
|-
|-
| BL_UHRR_||UHRR/ERCC||100pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id||totoRNAseq||
| BL_HuNu_||HuNu/ERCC||1ng - RnaseIII Fragmented 10 min||TSO.r06||T20V.id1||P1-STRT/N2_id25||15 Cycles||totoRNAseq||||
|-
|-
| BL_UHRR_||UHRR/ERCC||10pg - RNaseIII Fragmented - 1 step||TSO_r03||T20V.id||N2_id||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||||
 
== 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'''
 
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|2ng/ul, 200ng/ul, 20ng/ul UHRR||||||||||0.5ul
|-
|-
|ERCC 1:10E4, 1:10E5, 1:10E6||||||||||0.2ul
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id28||15 Cycles||totoRNAseq||||
|-
|Dilution Buffer||||||||||2.375ul
|-
|3' SMART CDS Primer  II A (12uM)||||||||||1.0ul
|-
|RNase Inhibitor||||||||||0.125ul
|-
|Total||||||||||4.5ul
|-
|-
|}
|}




:::''Incubate @ 72C 3 min''
[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List Primer List]
:::''Transfer to ice''


UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = Human Pyramidal Nuclei sorted 09-23-13


::{| {{table}}
== Purpose ==
|-
|||||||||||<u>Volume</u>
|-
|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'''
 
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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'''
 
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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'''
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|PAP RNA ||||||||||2.5ul
|-
|10mM dNTP Mix||||||||||1ul
|-
|12uM CDS II A Primer||||||||||1ul
|-
|dH2O||||||||||2ul
|-
|}
 
 
 
:::''Incubate @ 72C 3 min''
:::''Transfer to Ice''
 
 
 
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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'''
 
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|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'''
 
::{| {{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
|-
|}
 


*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 ==
== RNAseIII-Fragmented Library Preparation ==
Line 476: Line 82:
|10x RNaseIII Buffer||||||||||0.2ul
|10x RNaseIII Buffer||||||||||0.2ul
|-
|-
|1uM T20V.id1 ||||||||||0.1ul
|1uM or 0.1uM (10-100pg) T20V.id1 ||||||||||0.1ul
|-
|-
|dH20||||||||||0.25ul
|dH20||||||||||0.25ul
Line 505: Line 111:




:::''Incubate @ 37C 10 min''
:::''Incubate @ 37C 5-10 min''


:::''Add 1.1ul dH2O
:::''Add 1.1ul dH2O
Line 519: Line 125:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|PNK_fRNA||||||||||3.1ul
|fRNA||||||||||3.1ul
|-
|-
|5x RT Buffer||||||||||0.5ul
|5x RT Buffer||||||||||0.5ul
Line 545: Line 151:
|10mM dNTP Mix||||||||||1ul
|10mM dNTP Mix||||||||||1ul
|-
|-
|1uM T20V.ID ||||||||||0.9ul
|1uM or 0.1uM (10-100pg) T20V.ID ||||||||||0.9ul
|-
|-
|}
|}
Line 580: Line 186:
:'''Beads Purification'''
:'''Beads Purification'''


::#Add 25ul beads per well
::#Add 15ul beads per well
::#Allow to bind 15 min
::#Allow to bind 15 min
::#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 (x2)'''
:'''TSO'''


::{| {{table}}
::{| {{table}}
Line 636: Line 242:
|2x Buffer||||||||||25ul
|2x Buffer||||||||||25ul
|-
|-
|10uM P1-STRT (Smarter TSO)||||||||||1ul
|10uM P1-STRT (Smarter TSO) or PB_PCR_F||||||||||1ul
|-
|-
|10uM PCR_R_N2_id ||||||||||1ul
|10uM PCR_R_N2_id||||||||||1ul
|-
|-
|H2O||||||||||12ul
|H2O||||||||||12ul
Line 653: Line 259:
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|-
|98C 10s -> 72C 20s||||||||||6x (1ng), 9x (100pg), 12x (10pg)
|98C 10s -> 72C 20s||||||||||9x (1ng), 12x (100pg), 18x (10pg)
|-
|-
|72C, 5min
|72C, 5min

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


Primer List

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
  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 - 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

Final Library Preparation[edit]