Blue:RNA-Seq Experiments:totoRNAseq optimizations: Difference between revisions

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== Experimental Overview ==
== Experimental Overview ==


[[File:TotoRNAseq expanded optimization.jpg|600px]]
[[File:TotoRNAseq expanded optimization3.jpg|600px]]


== Purpose ==
== Purpose ==
Line 7: Line 7:
*Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries
*Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries
*Comparison of Mg-based and RNaseIII based fragmentation methods
*Comparison of Mg-based and RNaseIII based fragmentation methods
*Comparison of Enzymatics and Affymetrix PAP


== RNA-Seq Library Information ==
== RNA-Seq Library Information ==
Line 14: Line 13:
{| {{table}}
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Library ID'''
| align="center" style="background:#f0f0f0;"|'''Library ID'''
| align="center" style="background:#f0f0f0;"|'''Samples'''
| align="center" style="background:#f0f0f0;"|'''Sample'''
| align="center" style="background:#f0f0f0;"|'''Input RNA'''
| align="center" style="background:#f0f0f0;"|'''Condition'''
| align="center" style="background:#f0f0f0;"|'''TSO Primer'''
| align="center" style="background:#f0f0f0;"|'''5' Adaptor'''
| align="center" style="background:#f0f0f0;"|'''Bar Code Primers'''
| align="center" style="background:#f0f0f0;"|'''3' Adaptor'''
| align="center" style="background:#f0f0f0;"|'''Bar Code Primer 2'''
| align="center" style="background:#f0f0f0;"|'''N2 PCR Rev Primer'''
| align="center" style="background:#f0f0f0;"|'''Type of Seq'''
| align="center" style="background:#f0f0f0;"|'''RNA-Seq Protocol'''
| align="center" style="background:#f0f0f0;"|'''Reads'''
| align="center" style="background:#f0f0f0;"|'''Library Conc'''
|-
|-
| BL_totoRNAseq_1ngUHRR_||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06||T20VN_id1-4||N2.id1||totoRNAseq||||
| BL_totoRNAseq_1ngUHRR_||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1||N2.id1-2||totoRNAseq
|-
|-
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06||T20VN_id5-8||N2.id2||totoRNAseq||||
| ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1||N2.id3-4||totoRNAseq
|-
|-
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - STRT-Seq||TSO.r06||T20VN_id9-18||N2.id3||totoRNAseq||||
| ||10x10pg UHRR (combined)||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1-10||N2.id5||totoRNAseq
|-
|-
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06||T20VN_id1-4||N2.id4||totoRNAseq||||
| ||NTC||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1||N2.id6||totoRNAseq
|-
|-
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06||T20VN_id5-8||N2.id5||totoRNAseq||||
| ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||TSO.r06||T30VN_id1||N2.id7-8||totoRNAseq
|-
|-
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - STRT-Seq||TSO.r06||T20VN_id9-18||N2.id6||totoRNAseq||||
| ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||TSO.r06||T30VN_id1||N2.id9-10||totoRNAseq
|-
|-
| ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx||T20VN_id1-4||N2.id7||totoRNAseq||||
| ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 4 min||TSO.r06||T30VN_id1-10||N2.id11||totoRNAseq
|-
|-
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx||T20VN_id5-8||N2.id8||totoRNAseq||||
| ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min ||TSO.r06||T30VN_id1||N2.id12||totoRNAseq
|-
|-
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Quartz-Seq||xxx||T20VN_id9-18||N2.id9||totoRNAseq||||
| ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx||T20VN_id1-4||N2.id10||totoRNAseq||||
| ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx||T20VN_id5-8||N2.id11||totoRNAseq||||
| ||3x10pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x|||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Quartz-Seq||xxx||T20VN_id9-18||N2.id12||totoRNAseq||||
| ||NTC||2min Mg Frag/Enzymatics PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx||T20VN_id1-4||N2.id13||totoRNAseq||||
| ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx||T20VN_id5-8||N2.id14||totoRNAseq||||
| ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Cel-Seq||xxx||T20VN_id9-18||N2.id15||totoRNAseq||||
| ||3x10pg UHRR||0.05U RNase3 Frag/Affymetrix PAP 5 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx||T20VN_id1-4||N2.id16||totoRNAseq||||
| ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq
|-
|-
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx||T20VN_id5-8||N2.id17||totoRNAseq||||
| ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
|-
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Cel-Seq||xxx||T20VN_id9-18||N2.id18||totoRNAseq||||
| ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
|-
| ||UHRR/ERCC||4x1ng - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx||T20VN_id1-4||N2.id19||totoRNAseq||||
| ||3x10pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
|-
| ||UHRR/ERCC||4x100pg - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx||T20VN_id5-8||N2.id20||totoRNAseq||||
| ||NTC||2min Mg Frag/Enzymatics PAP 5 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
|-
| ||UHRR/ERCC||10x10pg - Mg Frag/Enzymatics PAP 5 min - Nugen||xxx||T20VN_id9-18||N2.id21||totoRNAseq||||
| ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
|-
| ||UHRR/ERCC||4x1ng - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx||T20VN_id1-4||N2.id22||totoRNAseq||||
| ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
|-
| ||UHRR/ERCC||4x100pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx||T20VN_id5-8||N2.id23||totoRNAseq||||
| ||3x10pg UHRR||0.05U RNase3 Frag/Affymetrix PAP 4 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
|-
| ||UHRR/ERCC||10x10pg - RNase3 Frag/Affymetrix PAP 10 min - Nugen||xxx||T20VN_id9-18||N2.id24||totoRNAseq||||
| ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||Smart2_TSO||Smart2_T30VN||N7xx/N5xx||totoRNAseq/Smart-Seq2
|-
| ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq
|-
| ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq
|-
| ||3x10pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression|||totoRNAseq/Quartz-Seq
|-
| ||NTC||2min Mg Frag/Enzymatics PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq
|-
| ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq
|-
| ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq
|-
| ||3x10pg UHRR||0.05U RNase3 Frag/Affymetrix PAP 5 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq
|-
| ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||Quartz_Tagging||Quartz-RT||Quartz-Suppression||totoRNAseq/Quartz-Seq
|-
| ||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id01-2||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq
|-
| ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id03-4||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq
|-
| ||10x10pg UHRR (combined)||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id05||MARS_T20N_id01-10||n/a|||totoRNAseq/MARS-Seq
|-
| ||NTC||2min Mg Frag/Enzymatics PAP 5 min||Lig_NNNX4_id06||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq
|-
| ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Lig_NNNX4_id07-8||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq
|-
| ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Lig_NNNX4_id9-10||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq
|-
| ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 5 min||Lig_NNNX4_id11||MARS_T20N_id01-10||n/a||totoRNAseq/MARS-Seq
|-
| ||NTC||0.1U RNase3 Frag/Affymetrix PAP 10 min||Lig_NNNX4_id12||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq
|-
|-
|}
|}
Line 75: Line 104:


Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]
Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence]
[[Blue:totoRNAseq Protocol Schemes|totoRNAseq Protocol Schemes]]


== cDNA Preparation: RNAseIII-Based Fragmentation ==
== cDNA Preparation: RNAseIII-Based Fragmentation ==
Line 85: Line 117:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|2ng/ul ||||||||||0.5ul
|2ng/ul or 0.2ng/ul or 0.02ng/ul ||||||||||0.5ul
|-
|-
|1:104  ERCC ||||||||||0.2ul
|1:10E4 or 1:10E5 or 1:10E6 ERCC ||||||||||0.2ul
|-
|-
|0.1uM T20VN||||||||||0.1ul
|0.1uM T30VN||||||||||0.1ul
|-
|-
|dH2O||||||||||0.2ul
|dH2O||||||||||0.2ul
Line 134: Line 166:
|1mM ATP||||||||||0.5ul   
|1mM ATP||||||||||0.5ul   
|-
|-
|0.5U/ul Enzymatics PAP (1:10d in H2O)||||||||||0.5ul + 0.3ul dH2O
|600U/ul Affymetrix PAP||||||||||0.2ul 
|-
|-
|or 600U/ul Affymetrix PAP||||||||||0.2ul + 0.6ul dH2O 
|40U/ul Rnase Inhibitor||||||||||0.2ul
|-
|-
|40U/ul Rnase Inhibitor||||||||||0.2ul
|dH2O||||||||||0.6ul
|-
|-
|Total||||||||||4ul  
|Total||||||||||4ul  
Line 145: Line 177:




:::''Incubate @ 37C 5 min (Enzymatics) or 4-10 min (Affymetrix)''
:::''Incubate @ 37C 4-10 min''


:::''Add 1ul of 0.1uM T20VN.id''
:::''Add 1ul of 0.1uM T30VN.id [totoRNAseq]''
:::''Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]''
:::''Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]''
:::''Add 1ul of 2uM MARS_T20N_id [MARS-Seq]''
:::''Add 1ul of ?uM ? [Nugen Ovation]''




Line 176: Line 212:
:::''Incubate @ 42C 30 min''
:::''Incubate @ 42C 30 min''


:::''For MARS-seq - continue to that section''


:'''Beads Purification'''
:'''Beads Purification'''


::#Add 15ul beads per well
::#Add 15ul beads per well
::#Allow to bind 15 min, combine wells
::#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
Line 193: Line 230:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|2ng/ul UHRR RNA||||||||||0.5ul
|2ng/ul or 0.2ng/ul or 0.02ng/ul ||||||||||0.5ul
|-
|-
|1:104 ERCC||||||||||0.2ul  
|1:10E4 or 1:10E5 or 1:10E6 ERCC ||||||||||0.2ul
|-
|-
|5x RT buffer||||||||||1ul
|5x RT buffer||||||||||1ul
Line 246: Line 283:
|0.5U/ul Enzymatics PAP (1:10d in H2O)||||||||||0.5ul
|0.5U/ul Enzymatics PAP (1:10d in H2O)||||||||||0.5ul
|-
|-
|or 600U/ul Affymetrix PAP||||||||||0.2ul + 0.3ul dH2O
|Total|||||||||| 4.1
|-
|-
|}
|}




:::''Incubate @ 37C 5 min (Enzymatics) or 10 min (Affymetrix)''
:::''Incubate @ 37C 5 min''
:::''Transfer to Ice''
:::''Transfer to Ice''


:::''Add 1ul of 0.1uM T20VN.id''
:::''Add 1ul of 0.1uM T30VN.id''




Line 278: Line 315:
|dH2O||||||||||0.5ul
|dH2O||||||||||0.5ul
|-
|-
|Total||||||||||10ul
|Total||||||||||10.1ul
|-
|-
|}
|}
Line 285: Line 322:
:::''Incubate @ 42C 30 min''
:::''Incubate @ 42C 30 min''


:::''For MARS-seq - continue to that section''


:'''Beads Purification'''
:'''Beads Purification'''


::#Add 15ul beads per well
::#Add 15ul beads per well
::#Allow to bind 15 min, combine wells
::#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; vacuspin ~8min to 3ul
::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul


== STRT-Seq Library Preparation ==
== totoRNASeq Library Preparation ==




Line 324: Line 362:




:::''Incubate @42C 10min''
:::''Incubate @42C 20min''
:::''Incubate @70C 10min''
:::''Incubate @70C 10min''


Line 355: Line 393:
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|-
|98C 10s -> 72C 20s||||||||||9x (1ng)
|98C 10s -> 72C 20s||||||||||9x (1ng); 12x (100pg)
|-
|-
|72C, 5min
|72C, 5min
Line 370: Line 408:
Results:
Results:


== Quartz-Seq Library Preparation ==
== UMI-STRT-Seq Library Preparation ==


'''Primer Digestion'''


:'''TSO Reaction'''


::{| {{table}}
::{| {{table}}
Line 381: Line 419:
|Purified cDNA||||||||||3ul
|Purified cDNA||||||||||3ul
|-
|-
|10x PCR Buffer||||||||||0.4ul
|5x First Strand buffer||||||||||1.4ul
|-
|10mM dNTP mix||||||||||0.7ul
|-
|100mM DTT||||||||||0.175ul
|-
|-
|10x Exo I buffer||||||||||0.2ul
|10uM C1-P1-RNA-TSO||||||||||0.7ul
|-
|-
|100mM DTT||||||||||0.2ul
|RNase Inhibitor||||||||||0.175ul
|-
|-
|Exonuclease I||||||||||0.6ul
|SuperScriptII||||||||||0.7ul
|-
|-
|dH2O||||||||||1.6ul
|dH2O||||||||||0.15ul
|-
|-
|Total||||||||||6ul
|Total||||||||||7ul
|-
|-
|}
|}




:::''Incubate @37C 30min''
:::''Incubate @42C 20min''
:::''Incubate @80C 20min''
:::''Incubate @70C 10min''
 




'''Poly-A Tailing'''
:'''PCR (Advantage)'''


::{| {{table}}
::{| {{table}}
Line 407: Line 448:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|ExoI RXN||||||||||6ul
|TSO Reaction||||||||||7ul
|-
|10x Advantage 2 Buffer||||||||||7ul
|-
|12uM C1-P1-PCR-2||||||||||2.8ul
|-
|dNTPs||||||||||2.8ul
|-
|Advantage 2 Polymerase||||||||||2.8ul
|-
|H2O||||||||||47.6ul
|-
|-
|10x PCR Buffer||||||||||0.5ul
|Total||||||||||70ul
|-
|-
|100mM dATP||||||||||0.15ul
|}
 
 
::{| {{table}}
|-
|-
|RNaseH||||||||||0.12ul
|95C 60 sec||||||||||
|-
|-
|TdT enzyme||||||||||0.42ul
|95C 20s -> 58C 4min -> 68C 6min||||||||||5x
|-
|-
|dH2O||||||||||3.81ul
|95C 20s -> 64C 30s -> 68C 1min||||||||||10x (1ng); 13x (100pg); 16x (10pg)
|-
|-
|Total||||||||||11ul
|72C, 10min
|-
|-
|}
|}




:::''Incubate @37C 50 sec''
 
:::''Incubate @65C 10min''
*Run 5ul on gel:
 
 
 
 
Results:
 




== Smart-Seq2 Library Preparation ==


'''Second Strand Synthesis'''


:'''TSO Reaction'''


::{| {{table}}
::{| {{table}}
Line 436: Line 497:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|Poly-A Tailed Sample||||||||||11ul
|Purified cDNA||||||||||3ul
|-
|5x First Strand buffer||||||||||2ul
|-
|10mM dNTP mix||||||||||1ul
|-
|100mM DTT||||||||||0.5ul
|-
|100uM Smart2-TSO||||||||||0.1ul
|-
|MgCl2 (1M)||||||||||0.06ul
|-
|Betaine (5M)||||||||||2ul
|-
|-
|2x PhusionHF Flex||||||||||25ul
|RNase Inhibitor||||||||||0.25ul
|-
|-
|10uM Smarter Tagging primer||||||||||0.32ul
|SuperScriptII||||||||||0.5ul
|-
|-
|dH2O||||||||||20.68ul
|dH2O||||||||||0.59ul
|-
|-
|Total||||||||||57ul
|Total||||||||||10ul
|-
|-
|}
|}


:::''Incubate @98C 30sec''
 
:::''Incubate @40C 1min''
:::''Incubate @42C 20min''
:::''Incubate @68C 5min''
:::''Incubate [@50C 2min > 42C 2min] x10 cycles''
:::''Incubate @70C 10min''
:::''Incubate @10C hold''




'''PCR Amplification'''
:'''PCR (KAPA-HiFi)'''


::{| {{table}}
::{| {{table}}
Line 459: Line 534:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|2nd Strand Sample||||||||||57ul
|TSO Reaction||||||||||10ul
|-
|2x PhusionHF Flex||||||||||25ul
|-
|-
|100uM P1-STRT||||||||||1ul
|2x KAPA HiFi Hotstart ReadyMix||||||||||12.5ul
|-
|-
|100uM PCR_R_N2.id||||||||||1ul
|IS PCR Primer (10uM)||||||||||0.25ul
|-
|-
|dH2O||||||||||23ul
|H2O||||||||||2.25ul
|-
|-
|Total||||||||||107ul
|Total||||||||||25ul
|-
|-
|}
|}
Line 476: Line 549:
::{| {{table}}
::{| {{table}}
|-
|-
|98C 30 sec||||||||||
|98C 3min||||||||||
|-
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|-
|98C 10s -> 72C 20s||||||||||9x (1ng) or 12x (100pg)
|98C 20s -> 67C 15s -> 72C 1min||||||||||15x (1ng); 18x (100pg); 21x (10pg)
|-
|-
|72C, 5min
|72C, 5min
Line 486: Line 557:
|}
|}


== Cel-Seq Library Preparation ==
== Quartz-Seq Library Preparation ==
 


'''Second Strand Synthesis'''
'''RT Primer Removal'''




Line 498: Line 568:
|Purified cDNA||||||||||3ul
|Purified cDNA||||||||||3ul
|-
|-
|FS Buffer||||||||||0.2ul
|10x PCR Buffer||||||||||0.4ul
|-
|-
|SS Buffer||||||||||1ul
|10x Exo I buffer||||||||||0.2ul
|-
|-
|10mM dNTP||||||||||0.8ul
|100mM DTT||||||||||0.2ul
|-
|-
|DNA Polymerase||||||||||0.2ul
|Exonuclease I||||||||||0.6ul
|-
|-
|RNaseH||||||||||0.1ul
|dH2O||||||||||1.6ul
|-
|-
|dH2O||||||||||4.7ul
|Total||||||||||6ul
|-
|Total||||||||||10ul
|-
|-
|}
|}


''Note: can add directly to beads and transfer to new tube''


:::''Incubate @16C 2 hours''
:::''Incubate @37C 30min''
:::''Incubate @80C 20min''
:::''Incubate @4C''




'''T7 Primer Addition'''


'''Poly-A Tailing'''


::{| {{table}}
::{| {{table}}
Line 525: Line 596:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|Second Strand Sample||||||||||10ul
|ExoI RXN||||||||||6ul
|-
|10x PCR Buffer||||||||||0.5ul
|-
|100mM dATP||||||||||0.15ul
|-
|-
|KAPA qPCR MM||||||||||25ul
|RNaseH||||||||||0.12ul
|-
|-
|RL.Add.T7 Primer (10uM)||||||||||0.5ul
|TdT enzyme||||||||||0.42ul
|-
|-
|dH2O||||||||||14.5ul
|dH2O||||||||||3.81ul
|-
|-
|Total||||||||||50ul
|Total||||||||||11ul
|-
|-
|}
|}


:::''Incubate @94C 2min''
:::''Incubate @60C 2min''
:::''Incubate @72C 5min''


'''Beads Purification'''
:::''Incubate @37C 50 sec''
:::''Incubate @65C 10min''
:::''Incubate @4C''
 


::#Add 50ul beads per well
::#Allow to bind 15 min, combine wells
::#Remove supernatant and wash 2x with 1ml 80% EtOH
::#Air Dry 10min or until cracking first occurs
::#Resuspend beads in 6.4ul dH2O, incubate 2min, transfer to new tube


'''Second Strand Synthesis'''


'''IVT'''


::{| {{table}}
::{| {{table}}
Line 556: Line 626:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|Purified T7-added Sample||||||||||6.4ul
|Poly-A Tailed Sample||||||||||11ul
|-
|-
|ATP||||||||||1.6ul
|2x Terra Direct PCR Buffer||||||||||25ul
|-
|-
|GTP||||||||||1.6ul
|10uM Tagging Primer||||||||||0.32ul
|-
|-
|CTP||||||||||1.6ul
|Terra Polymerase||||||||||2ul
|-
|-
|UTP||||||||||1.6ul
|dH2O||||||||||18.68ul
|-
|-
|10x T7 buffer||||||||||1.6ul
|Total||||||||||57ul
|-
|T7 Enzyme||||||||||1.6ul
|-
|Total||||||||||16ul
|-
|-
|}
|}


:::''Incubate @37C (lid 70C) 13 hours''
:::''Incubate @98C 2min 10 sec''
:::''Incubate @4C''
:::''Incubate @40C 1min''
:::''Incubate @68C 5min''
:::''Transfer to ice''


'''Quantification of aRNA'''


:*Determine concentration of amplified RNA using Qubit
'''PCR Amplification'''
:*Dilute 5-10ng RNA into 16ul dH2O
 
'''Phosphatase Treatment'''


::{| {{table}}
::{| {{table}}
Line 588: Line 652:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|aRNA||||||||||16ul
|2nd Strand Sample||||||||||57ul
|-
|-
|10x Phosphatase buffer||||||||||2ul
|2x Terra Direct PCR Buffer||||||||||25ul
|-
|-
|Anarctic phosphatase||||||||||1ul
|100uM PCR Primer||||||||||1ul
|-
|-
|RNase Inhibitor||||||||||1ul
|dH2O||||||||||24ul
|-
|-
|Total||||||||||20ul
|Total||||||||||107ul
|-
|-
|}
|}


:::''Incubate @37C 30min''
:::''Incubate @65C 5min''
:::''Incubate @4C Indefinitely''
'''PNK Treatment'''


::{| {{table}}
::{| {{table}}
|-
|-
|||||||||||<u>Volume</u>
|68C 1 sec||||||||||
|-
|Phos Treated aRNA||||||||||20ul
|-
|dH2O||||||||||17ul
|-
|-
|10x phosphatase buffer||||||||||5ul
|98C 10s -> 65C 15s -> 68C 5min||||||||||15x (1ng); 18x (100pg); 21x (10pg)
|-
|-
|ATP (10mM, from Illumina kit)||||||||||5ul
|68C, 5min
|-
|RNase Inhibitor||||||||||1ul
|-
|PNK||||||||||2ul
|-
|Total||||||||||50ul
|-
|-
|}
|}


:::''Incubate @37C 60min''
== MARS-Seq Library Preparation ==
 
'''Exonuclease Digestion'''
 
:::''Add 1ul Exonuclease I (1:10 diluted in 1x exonuclease buffer)to each RT reaction''


'''RNA Cleanup: (Zymo RNA Clean and Concentrator-5)'''
:::''Incubate at 37C for 30min''
:::''Incubate at 80C for 10min''


:*Add 100ul RNA Binding Buffer and mix well
:*Add 150ul 95-100% Ethanol and mix well
:*Transfer to Zymo-Spin IC Column and centrifuge 1min
:*Add 400ul RNA Prep Buffer to column and centrifuge 1min
:*Add 800ul RNA Wash Buffer to column and centrifuge 1min
:*Add 400ul RNA Wash Buffer to column and centrifuge 2min
:*Transfer column to 1.5ml tube, add 6ul dH2O, 1min RT, centrifuge 1min
:*Speedvac to 5ul


:''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 17ul 10mM Tris-HCl pH7.5, incubate 2min, transfer to new tube


'''3'Adaptor Ligation'''
 
'''Second Strand Synthesis'''


::{| {{table}}
::{| {{table}}
Line 646: Line 699:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|Phos/PNK aRNA||||||||||5ul
|cDNA||||||||||17ul
|-
|10x NEB second strand synthesis buffer||||||||||2ul
|-
|Second strand synthesis enzyme||||||||||1ul
|-
|-
|3' Smarter Adaptor RA3 (1:5 dilution?)||||||||||1ul
|Total||||||||||20ul
|-
|-
|}
|}


:::''Incubate @70C 2min''
 
:::''transfer to ice''
:::''Incubate @ 16C for 2.5 hours''
 
 
'''IVT'''
 
 
::#Add 28ul 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:
 


::{| {{table}}
::{| {{table}}
Line 659: Line 727:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|aRNA/adaptor||||||||||6ul
|10x Reaction Buffer (T7 high yield NEB)||||||||||2ul
|-
|-
|5x HM Ligation Buffer (HML, Illumina kit)||||||||||2ul
|UTP||||||||||2ul
|-
|-
|RNase Inhibitor||||||||||1ul
|ATP||||||||||2ul
|-
|-
|T4 RNA Ligase 2, truncated||||||||||1ul
|CTP||||||||||2ul
|-
|GTP||||||||||2ul
|-
|T7 polymerase||||||||||2ul
|-
|dH2O||||||||||8ul
|-
|Total||||||||||20ul
|-
|-
|}
|}


:::''Incubate @28C 1 hour (unheated lid)''
:::''Incubate 16 hours (overnight) @ 37C'''
:::''Still in thermocycler: add 1ul stop solution (STP)''
:::''Add 1ul Turbo DNase I and incubate @ 37C 15min
:::''Pipette entire volume 6-8 times to mix''
 
:::''Incubate @28C 15min''
 
:::''Transfer to ice''
:''Beads Purification:''
 
::#Add 24ul 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 dH20, transfer to new tube, vacuspin to 3ul


:::''Add 3ul dH2O''




'''Reverse Transcription'''
'''Adaptor Ligation'''


::{| {{table}}
::{| {{table}}
Line 684: Line 765:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|Adaptor-ligated RNA||||||||||6ul (store remaining at -80C)
|aRNA||||||||||3ul
|-
|-
|RNA Smarter RT Primer (RTP)||||||||||1ul
|100uM Lig_NNNX4_idx||||||||||1ul
|-
|-
|}
|}


:::''Incubate @70C 2min''
:::''Incubate @ 70C for 3min''
:::''Immediately transfer to ice''
 


::{| {{table}}
::{| {{table}}
Line 697: Line 778:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|RNA/primer||||||||||7ul
|aRNA/adaptor||||||||||4ul
|-
|-
|10x RT Buffer||||||||||1ul
|10x T4 Ligase buffer||||||||||1ul
|-
|-
|10mM dNTP||||||||||0.5ul
|DMSO||||||||||0.95ul
|-
|-
|100mM DTT||||||||||0.5ul
|10mM ATP||||||||||1ul
|-
|PEG8000||||||||||2ul
|-
|-
|RNase Inhibitor||||||||||0.5ul
|T4 RNA Ligase I(10U/ul)||||||||||1ul
|-
|-
|SuperScriptIII||||||||||0.5ul
|dH2O||||||||||0ul
|-
|-
|Total||||||||||10ul
|Total||||||||||10ul
Line 713: Line 796:
|}
|}


:::''Incubate at 22C for 2hrs''


:::''Incubate @25C 5min''
:::''Incubate @50C 20min''
:::''Incubate @70C 10min''


 
'''Reverse Transcription'''
'''PCR Amplification'''


::{| {{table}}
::{| {{table}}
Line 725: Line 805:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|RT Reaction||||||||||10ul
|Ligated RNA||||||||||8ul
|-
|-
|2x PhusionHF Flex Buffer||||||||||25ul
|10uM MARS_RT_2||||||||||1ul
|-
|-
|10uM P1-STRT||||||||||1ul
|10mM dNTP||||||||||1ul
|-
|-
|10uM PCR_R_N2_id||||||||||1ul
|Total||||||||||10ul
|-
|H2O||||||||||13ul
|-
|Total||||||||||50ul
|-
|-
|}
|}




::{| {{table}}
:::''Incubate @ 65C 5min''
|-
:::''Incubate on ice 1min''
|98C 30 sec||||||||||
|-
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|98C 10s -> 72C 20s||||||||||9x (10ng)
|-
|72C, 5min
|-
|}
 
 
== Nugen Ovation Library Preparation ==
 
 
'''Second Strand Synthesis'''




Line 763: Line 824:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|cDNA||||||||||3ul
|RNA/Primer||||||||||10ul
|-
|-
|2x PhusionHF Flex||||||||||5ul
|10x RT Buffer||||||||||2ul
|-
|-
|10uM Smarter random primer||||||||||0.3ul
|25mM MgCl2||||||||||0.8ul
|-
|-
|RNaseH||||||||||0.1ul
|100mM DTT||||||||||2ul
|-
|-
|dH2O||||||||||1.6ul
|RNase Inhibitor (40U/ul)||||||||||1ul
|-
|SSIII (200U/ul)||||||||||1ul
|-
|dH2O||||||||||3.2ul
|-
|-
|Total||||||||||10ul
|Total||||||||||10ul
Line 777: Line 842:
|}
|}


:::''Incubate @37C 20min''
 
:::''Incubate @98C 30sec''
:::''Incubate @42C 10min''
:::''Incubate @25C 30min''
:::''Incubate @50C 50min''
:::''Incubate @70C 10min''
:::''Incubate @85C 5min''
 
 
'''Beads Purify'''
 
::#Add 30ul 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 dH20, transfer to new tube




Line 789: Line 863:
|||||||||||<u>Volume</u>
|||||||||||<u>Volume</u>
|-
|-
|2nd Strand Sample||||||||||10ul
|cDNA||||||||||10ul
|-
|-
|2x PhusionHF Flex||||||||||50ul
|SybrGreen KAPA 2x MM||||||||||25ul
|-
|-
|100uM P1-STRT||||||||||1ul
|10uM P5_Rd1_PCR_F||||||||||2.5ul
|-
|-
|100uM PCR_R_N2.id||||||||||1ul
|10uM P7_Rd2_PCR_R||||||||||2.5ul
|-
|-
|dH2O||||||||||38ul
|dH2O||||||||||
|-
|Total||||||||||100ul
|-
|-
|}
|}
Line 806: Line 878:
::{| {{table}}
::{| {{table}}
|-
|-
|98C 30 sec||||||||||
|95C 30 sec||||||||||
|-
|98C 15s -> 58C 20s -> 72C 30s||||||||||6x
|-
|-
|98C 10s -> 72C 20s||||||||||9x (1ng) or 12x (100pg)
|95C 15s -> 60C 20s -> 72C 30s||||||||||12x
|-
|-
|72C, 5min
|72C, 10min
|-
|-
|}
|}
== Nextera XT for Quartz-Seq ==

Latest revision as of 01:27, 22 February 2014

Experimental Overview[edit]

File:TotoRNAseq expanded optimization3.jpg

Purpose[edit]

  • Systematic analysis of different methods for addition of forward ILMN sequencing adapter to totoRNAseq libraries
  • Comparison of Mg-based and RNaseIII based fragmentation methods

RNA-Seq Library Information[edit]

Library ID Sample Condition 5' Adaptor 3' Adaptor N2 PCR Rev Primer RNA-Seq Protocol
BL_totoRNAseq_1ngUHRR_ 2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1 N2.id1-2 totoRNAseq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1 N2.id3-4 totoRNAseq
10x10pg UHRR (combined) 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1-10 N2.id5 totoRNAseq
NTC 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1 N2.id6 totoRNAseq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id7-8 totoRNAseq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id9-10 totoRNAseq
10x10pg UHRR (combined) 0.05U RNase3 Frag/Affymetrix PAP 4 min TSO.r06 T30VN_id1-10 N2.id11 totoRNAseq
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id12 totoRNAseq
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
3x10pg UHRR 2min Mg Frag/Enzymatics PAP 5 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
NTC 2min Mg Frag/Enzymatics PAP 5 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
3x10pg UHRR 0.05U RNase3 Frag/Affymetrix PAP 5 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min C1-P1-RNA-TSO C1-P1-T31 C1-TN5-x totoRNAseq/UMI-STRT-Seq
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
3x10pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
NTC 2min Mg Frag/Enzymatics PAP 5 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
3x10pg UHRR 0.05U RNase3 Frag/Affymetrix PAP 4 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min Smart2_TSO Smart2_T30VN N7xx/N5xx totoRNAseq/Smart-Seq2
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
3x10pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
NTC 2min Mg Frag/Enzymatics PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
3x10pg UHRR 0.05U RNase3 Frag/Affymetrix PAP 5 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min Lig_NNNX4_id01-2 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Lig_NNNX4_id03-4 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
10x10pg UHRR (combined) 2min Mg Frag/Enzymatics PAP 5 min Lig_NNNX4_id05 MARS_T20N_id01-10 n/a totoRNAseq/MARS-Seq
NTC 2min Mg Frag/Enzymatics PAP 5 min Lig_NNNX4_id06 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Lig_NNNX4_id07-8 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min Lig_NNNX4_id9-10 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
10x10pg UHRR (combined) 0.05U RNase3 Frag/Affymetrix PAP 5 min Lig_NNNX4_id11 MARS_T20N_id01-10 n/a totoRNAseq/MARS-Seq
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min Lig_NNNX4_id12 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq


Primer sequences [1]


totoRNAseq Protocol Schemes

cDNA Preparation: RNAseIII-Based Fragmentation[edit]

RNA
Volume
2ng/ul or 0.2ng/ul or 0.02ng/ul 0.5ul
1:10E4 or 1:10E5 or 1:10E6 ERCC 0.2ul
0.1uM T30VN 0.1ul
dH2O 0.2ul
Total 1ul


Incubate @ 72C 3 min
Incubate @ 37C 1 min


Volume
10x RNaseIII buffer 0.2ul
RNase III 0.1ul
dH20 0.7ul
Total 2ul


Incubate @ 37C 1 min


3' Tailing
Volume
fRNA 2ul
5x RT buffer 0.5ul
1mM ATP 0.5ul
600U/ul Affymetrix PAP 0.2ul
40U/ul Rnase Inhibitor 0.2ul
dH2O 0.6ul
Total 4ul


Incubate @ 37C 4-10 min
Add 1ul of 0.1uM T30VN.id [totoRNAseq]
Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]
Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]
Add 1ul of 2uM MARS_T20N_id [MARS-Seq]
Add 1ul of ?uM ? [Nugen Ovation]


Reverse Transcription
Volume
PAP RNA 5ul
5xRT Buffer 1.5ul
10mM dNTP Mix 1ul
100mM DTT 0.25ul
Betaine (5M) 2ul
Smarter MMLV (200U/ul) 0.25ul
Total 10ul


Incubate @ 42C 30 min
For MARS-seq - continue to that section
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 ~8min to 3ul

cDNA Preparation: Mg-based Fragmentation[edit]

RNA FRAGMENTATION
Volume
2ng/ul or 0.2ng/ul or 0.02ng/ul 0.5ul
1:10E4 or 1:10E5 or 1:10E6 ERCC 0.2ul
5x RT buffer 1ul
dH2O 0.3ul
Total 2ul


Incubate @ 94C 2 min
Transfer to ice


End Repair
Volume
fRNA 2ul
1mM ATP 0.4ul
100mM DTT 0.1ul
RNase Inhibitor 0.1ul
PNK 0.5ul
Total 3.1ul


Incubate @ 37C 30 min


3' Tailing
Volume
PNK_fRNA 3.1ul
1mM ATP 0.5ul
0.5U/ul Enzymatics PAP (1:10d in H2O) 0.5ul
Total 4.1


Incubate @ 37C 5 min
Transfer to Ice
Add 1ul of 0.1uM T30VN.id


Reverse Transcription
Volume
PAP RNA 5ul
5xRT Buffer 1ul
10mM dNTP Mix 1ul
100mM DTT 0.25ul
Betaine (5M) 2ul
Smarter MMLV (200U/ul) 0.25ul
dH2O 0.5ul
Total 10.1ul


Incubate @ 42C 30 min
For MARS-seq - continue to that section
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 ~8min to 3ul

totoRNASeq Library Preparation[edit]

TSO Reaction
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 20min
Incubate @70C 10min


PCR (PhusionHF)
Volume
TSO Reaction 10ul
2x Buffer 25ul
10uM P1-STRT 1ul
10uM PCR_R_N2_id 1ul
H2O 13ul
Total 50ul


98C 30 sec
98C 15s -> 58C 20s -> 72C 30s 6x
98C 10s -> 72C 20s 9x (1ng); 12x (100pg)
72C, 5min


  • Run 5ul on gel:



Results:

UMI-STRT-Seq Library Preparation[edit]

TSO Reaction
Volume
Purified cDNA 3ul
5x First Strand buffer 1.4ul
10mM dNTP mix 0.7ul
100mM DTT 0.175ul
10uM C1-P1-RNA-TSO 0.7ul
RNase Inhibitor 0.175ul
SuperScriptII 0.7ul
dH2O 0.15ul
Total 7ul


Incubate @42C 20min
Incubate @70C 10min


PCR (Advantage)
Volume
TSO Reaction 7ul
10x Advantage 2 Buffer 7ul
12uM C1-P1-PCR-2 2.8ul
dNTPs 2.8ul
Advantage 2 Polymerase 2.8ul
H2O 47.6ul
Total 70ul


95C 60 sec
95C 20s -> 58C 4min -> 68C 6min 5x
95C 20s -> 64C 30s -> 68C 1min 10x (1ng); 13x (100pg); 16x (10pg)
72C, 10min


  • Run 5ul on gel:



Results:


Smart-Seq2 Library Preparation[edit]

TSO Reaction
Volume
Purified cDNA 3ul
5x First Strand buffer 2ul
10mM dNTP mix 1ul
100mM DTT 0.5ul
100uM Smart2-TSO 0.1ul
MgCl2 (1M) 0.06ul
Betaine (5M) 2ul
RNase Inhibitor 0.25ul
SuperScriptII 0.5ul
dH2O 0.59ul
Total 10ul


Incubate @42C 20min
Incubate [@50C 2min > 42C 2min] x10 cycles
Incubate @70C 10min
Incubate @10C hold


PCR (KAPA-HiFi)
Volume
TSO Reaction 10ul
2x KAPA HiFi Hotstart ReadyMix 12.5ul
IS PCR Primer (10uM) 0.25ul
H2O 2.25ul
Total 25ul


98C 3min
98C 20s -> 67C 15s -> 72C 1min 15x (1ng); 18x (100pg); 21x (10pg)
72C, 5min

Quartz-Seq Library Preparation[edit]

RT Primer Removal


Volume
Purified cDNA 3ul
10x PCR Buffer 0.4ul
10x Exo I buffer 0.2ul
100mM DTT 0.2ul
Exonuclease I 0.6ul
dH2O 1.6ul
Total 6ul

Note: can add directly to beads and transfer to new tube

Incubate @37C 30min
Incubate @80C 20min
Incubate @4C


Poly-A Tailing

Volume
ExoI RXN 6ul
10x PCR Buffer 0.5ul
100mM dATP 0.15ul
RNaseH 0.12ul
TdT enzyme 0.42ul
dH2O 3.81ul
Total 11ul


Incubate @37C 50 sec
Incubate @65C 10min
Incubate @4C


Second Strand Synthesis


Volume
Poly-A Tailed Sample 11ul
2x Terra Direct PCR Buffer 25ul
10uM Tagging Primer 0.32ul
Terra Polymerase 2ul
dH2O 18.68ul
Total 57ul
Incubate @98C 2min 10 sec
Incubate @40C 1min
Incubate @68C 5min
Transfer to ice


PCR Amplification

Volume
2nd Strand Sample 57ul
2x Terra Direct PCR Buffer 25ul
100uM PCR Primer 1ul
dH2O 24ul
Total 107ul


68C 1 sec
98C 10s -> 65C 15s -> 68C 5min 15x (1ng); 18x (100pg); 21x (10pg)
68C, 5min

MARS-Seq Library Preparation[edit]

Exonuclease Digestion

Add 1ul Exonuclease I (1:10 diluted in 1x exonuclease buffer)to each RT reaction
Incubate at 37C for 30min
Incubate at 80C for 10min


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 17ul 10mM Tris-HCl pH7.5, incubate 2min, transfer to new tube


Second Strand Synthesis

Volume
cDNA 17ul
10x NEB second strand synthesis buffer 2ul
Second strand synthesis enzyme 1ul
Total 20ul


Incubate @ 16C for 2.5 hours


IVT


  1. Add 28ul 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:


Volume
10x Reaction Buffer (T7 high yield NEB) 2ul
UTP 2ul
ATP 2ul
CTP 2ul
GTP 2ul
T7 polymerase 2ul
dH2O 8ul
Total 20ul
Incubate 16 hours (overnight) @ 37C'
Add 1ul Turbo DNase I and incubate @ 37C 15min


Beads Purification:
  1. Add 24ul 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 10ul dH20, transfer to new tube, vacuspin to 3ul


Adaptor Ligation

Volume
aRNA 3ul
100uM Lig_NNNX4_idx 1ul
Incubate @ 70C for 3min


Volume
aRNA/adaptor 4ul
10x T4 Ligase buffer 1ul
DMSO 0.95ul
10mM ATP 1ul
PEG8000 2ul
T4 RNA Ligase I(10U/ul) 1ul
dH2O 0ul
Total 10ul
Incubate at 22C for 2hrs


Reverse Transcription

Volume
Ligated RNA 8ul
10uM MARS_RT_2 1ul
10mM dNTP 1ul
Total 10ul


Incubate @ 65C 5min
Incubate on ice 1min


Volume
RNA/Primer 10ul
10x RT Buffer 2ul
25mM MgCl2 0.8ul
100mM DTT 2ul
RNase Inhibitor (40U/ul) 1ul
SSIII (200U/ul) 1ul
dH2O 3.2ul
Total 10ul


Incubate @42C 10min
Incubate @50C 50min
Incubate @85C 5min


Beads Purify

  1. Add 30ul 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 10ul dH20, transfer to new tube


PCR Amplification

Volume
cDNA 10ul
SybrGreen KAPA 2x MM 25ul
10uM P5_Rd1_PCR_F 2.5ul
10uM P7_Rd2_PCR_R 2.5ul
dH2O


95C 30 sec
95C 15s -> 60C 20s -> 72C 30s 12x
72C, 10min

Nextera XT for Quartz-Seq[edit]