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

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| 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||SMART CDS ||||In-house Smarter||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||SMART CDS|||||In-house Smarter|||
| 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||SMART CDS||||In-house Smarter||||
| 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||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||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||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||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||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||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:
 
:'''RNA PREPARATION'''
 
::{| {{table}}
|-
|-
|||||||||||<u>Volume</u>
| BL_HuLCM_||HuLCM/ERCC||1ng - RnaseIII Fragmented 5 min||TSO.r06||T20V.id1||P1-STRT/N2_id28||15 Cycles||totoRNAseq||||
|-
|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''


[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List Primer List]


::{| {{table}}
UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = Human Pyramidal Nuclei sorted 09-23-13
|-
|||||||||||<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
|-
|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
|-
|}


== Purpose ==


 
*Generate totoRNAseq libraries using several TSO to compare efficiencies
:::''Incubate @ 72C 3 min''
*Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal nuclei
:::''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


== RNAseIII-Fragmented Library Preparation ==
== RNAseIII-Fragmented Library Preparation ==
Line 244: Line 82:
|10x RNaseIII Buffer||||||||||0.2ul
|10x RNaseIII Buffer||||||||||0.2ul
|-
|-
|1uM T20V.id1 or CDS II A||||||||||0.1ul
|1uM or 0.1uM (10-100pg) T20V.id1 ||||||||||0.1ul
|-
|-
|dH20||||||||||0.25ul
|dH20||||||||||0.25ul
Line 273: Line 111:




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


:::''Add 1.1ul dH2O
:::''Add 1.1ul dH2O
Line 287: 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 301: Line 139:
:::''Transfer to Ice''
:::''Transfer to Ice''


== Reverse Transcription: ==
 
 
:'''Reverse Transcription'''


::{| {{table}}
::{| {{table}}
Line 307: Line 147:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|PAP RNA or unfrag RNA (diluted to 3.6ul in dH2O)||||||||||4.1ul (3.6ul)
|PAP RNA ||||||||||4.1ul
|-
|-
|10mM dNTP Mix||||||||||1ul
|10mM dNTP Mix||||||||||1ul
|-
|-
|1uM T20V.ID or Supp.T20.id||||||||||0.9ul
|1uM or 0.1uM (10-100pg) T20V.ID ||||||||||0.9ul
|-
|-
|}
|}
Line 326: Line 166:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|5xRT Buffer||||||||||1.5ul (RNaseIII); 1ul + 0.5ul dH2O (Mg); 2ul (unfrag)
|5xRT Buffer||||||||||1.5ul  
|-
|-
|100mM DTT||||||||||0.17ul
|100mM DTT||||||||||0.17ul
Line 350: 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 (x2)'''
:'''TSO'''


::{| {{table}}
::{| {{table}}
Line 403: Line 242:
|2x Buffer||||||||||25ul
|2x Buffer||||||||||25ul
|-
|-
|10uM [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence PB_PCR_F]; P1-STRT (Smarter TSO); or PCR_Blue_Short||||||||||1ul
|10uM P1-STRT (Smarter TSO) or PB_PCR_F||||||||||1ul
|-
|-
|10uM PGR_PCR_R or PCR_Blue_Short||||||||||1ul
|10uM PCR_R_N2_id||||||||||1ul
|-
|-
|H2O||||||||||12ul
|H2O||||||||||12ul
Line 420: Line 259:
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|-
|98C 10s -> 72C 20s||||||||||11x
|98C 10s -> 72C 20s||||||||||9x (1ng), 12x (100pg), 18x (10pg)
|-
|-
|72C, 5min
|72C, 5min
Line 428: Line 267:
== Results of Amplification ==
== Results of Amplification ==


*Ran 5ul on gel:
*Ampure Beads Purify (0.8x)
 
*Resuspend in 10ul
[[File:09102013_PCR1_gel1.jpg]]
*Run 1ul on gel
 
== TSO Quantification ==
*Comparison of Pre-amp and post-amp plots:
::{| {{table}}
| align="center" style="background:#f0f0f0;"|'''PCR - 5' TSO'''
| align="center" style="background:#f0f0f0;"|'''PCR - 3' T20'''
| align="center" style="background:#f0f0f0;"|'''PCR fragment'''
|-
| TSO+130-5'||T20+130-5'||ERCC-130-5'
|-
| TSO+130-mid||T20+130-mid||ERCC-130-mid
|-
| TSO+130-3'||T20+130-3'||ERCC-130-3'
|-
|
|}
 
 
TSO=ILMN_PCR_F; T20=ILMN_PCR_R
 
 
*Amplified 1ul TSO (diluted 5ul to 50ul) and 1ul PCR (total volume 50ul) 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;"|'''RnaseIII'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Mg'''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|''''''
| align="center" style="background:#f0f0f0;"|'''Unfrag'''
| 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;"|TSO||r04||r04||r04||r04||r04||r04||r04||r04||r04
|-
| align="center" style="background:#f0f0f0;"|TSO||r05||r05||r05||r05||r05||r05||r05||r05||r05
|-
| align="center" style="background:#f0f0f0;"|TSO||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart
|-
| align="center" style="background:#f0f0f0;"|TSO||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp
|-
| align="center" style="background:#f0f0f0;"|PCR||r04||r04||r04||r04||r04||r04||r04||r04||r04
|-
| align="center" style="background:#f0f0f0;"|PCR||r05||r05||r05||r05||r05||r05||r05||r05||r05
|-
| align="center" style="background:#f0f0f0;"|PCR||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart||Smart
|-
| align="center" style="background:#f0f0f0;"|PCR||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp||r04+Supp
|-
|
|}
 
 
 
[[File:09102013_ERCC-R_PCR-F.jpg]]
 
[[File:09102013_ERCC130_Unfrag.jpg]]
 
[[File:09102013_ERCC130_RIII.jpg]]
 
[[File:09102013_ERCC130_Mg.jpg]]


== Final Library Preparation ==
== Final Library Preparation ==
*Beads Purified PCR product (0.8x) and resuspended in 10ul:
*Re-Amplified using PB_PCR_F or P1-STRT and PCR_R_N2_id primers:
:#r04 - RnaseIII - id9
:#r04 - Mg - id10
:#r05 - RnaseIII - id11
:#r05 - Mg - id12
:#Smart - RnaseIII - id13
:#Smart - Mg - id14
::{| {{table}}
|-
|||||||||||<u>Volume</u>
|-
|Library||||||||||1ul Smart or 4ul r04/r05
|-
|2xSybr Buffer||||||||||25ul
|-
|10uM F primer||||||||||1ul
|-
|10uM R Primer||||||||||1ul
|-
|dH2O||||||||||up to 50ul
|-
|}
:::''95C 30sec''
:::''95C 10s -> 60C 20s -> 72C 30s''  => 5X
:::''72C, 5min''
*Beads purified (0.8x), resuspended in 10ul and ran 1ul on gel:
[[File:09102013_PCR2_gel1.jpg]]

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]