Blue:RNA-Seq Experiments:totoRNAseq optimizations: Difference between revisions
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== Experimental Overview == | == Experimental Overview == | ||
[[File:TotoRNAseq expanded | [[File:TotoRNAseq expanded optimization3.jpg|600px]] | ||
== Purpose == | == Purpose == | ||
Line 20: | Line 20: | ||
| align="center" style="background:#f0f0f0;"|'''RNA-Seq Protocol''' | | align="center" style="background:#f0f0f0;"|'''RNA-Seq Protocol''' | ||
|- | |- | ||
| BL_totoRNAseq_1ngUHRR_||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T30VN_id1||N2.id1-2||totoRNAseq | | 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 | | ||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. | | ||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. | | ||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. | | ||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. | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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|| | | ||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 134: | Line 166: | ||
|1mM ATP||||||||||0.5ul | |1mM ATP||||||||||0.5ul | ||
|- | |- | ||
| | |600U/ul Affymetrix PAP||||||||||0.2ul | ||
|- | |- | ||
| | |40U/ul Rnase Inhibitor||||||||||0.2ul | ||
|- | |- | ||
| | |dH2O||||||||||0.6ul | ||
|- | |- | ||
|Total||||||||||4ul | |Total||||||||||4ul | ||
Line 145: | Line 177: | ||
:::''Incubate @ 37C | :::''Incubate @ 37C 4-10 min'' | ||
:::''Add 1ul of 0.1uM T30VN.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 | ::#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 | ::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul | ||
== cDNA Preparation: Mg-based Fragmentation == | == cDNA Preparation: Mg-based Fragmentation == | ||
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 | ||
|- | |- | ||
| | |Total|||||||||| 4.1 | ||
|- | |- | ||
|} | |} | ||
:::''Incubate @ 37C 5 min | :::''Incubate @ 37C 5 min'' | ||
:::''Transfer to Ice'' | :::''Transfer to Ice'' | ||
Line 278: | Line 315: | ||
|dH2O||||||||||0.5ul | |dH2O||||||||||0.5ul | ||
|- | |- | ||
|Total|||||||||| | |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 | ::#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 | ::#Resuspend beads in 6ul dH2O, incubate 2min, transfer to new tube; vacuspin ~8min to 3ul | ||
== | == totoRNASeq Library Preparation == | ||
Line 324: | Line 362: | ||
:::''Incubate @42C | :::''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: | ||
== | == UMI-STRT-Seq Library Preparation == | ||
:'''TSO Reaction''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 381: | Line 419: | ||
|Purified cDNA||||||||||3ul | |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|||||||||| | |dH2O||||||||||0.15ul | ||
|- | |- | ||
|Total|||||||||| | |Total||||||||||7ul | ||
|- | |- | ||
|} | |} | ||
:::''Incubate @ | :::''Incubate @42C 20min'' | ||
:::''Incubate @ | :::''Incubate @70C 10min'' | ||
''' | :'''PCR (Advantage)''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 407: | Line 448: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |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 | ||
|- | |- | ||
| | |} | ||
::{| {{table}} | |||
|- | |- | ||
| | |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 == | |||
:'''TSO Reaction''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 436: | Line 497: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |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|||||||||| | |dH2O||||||||||0.59ul | ||
|- | |- | ||
|Total|||||||||| | |Total||||||||||10ul | ||
|- | |- | ||
|} | |} | ||
:::''Incubate @ | |||
:::''Incubate @ | :::''Incubate @42C 20min'' | ||
:::''Incubate @ | :::''Incubate [@50C 2min > 42C 2min] x10 cycles'' | ||
:::''Incubate @70C 10min'' | |||
:::''Incubate @10C hold'' | |||
'''PCR | :'''PCR (KAPA-HiFi)''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 461: | Line 534: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |TSO Reaction||||||||||10ul | ||
|- | |- | ||
|2x | |2x KAPA HiFi Hotstart ReadyMix||||||||||12.5ul | ||
|- | |- | ||
| | |IS PCR Primer (10uM)||||||||||0.25ul | ||
|- | |- | ||
| | |H2O||||||||||2.25ul | ||
|- | |- | ||
|Total|||||||||| | |Total||||||||||25ul | ||
|- | |- | ||
|} | |} | ||
Line 476: | Line 549: | ||
::{| {{table}} | ::{| {{table}} | ||
|- | |- | ||
| | |98C 3min|||||||||| | ||
|- | |- | ||
|98C | |98C 20s -> 67C 15s -> 72C 1min||||||||||15x (1ng); 18x (100pg); 21x (10pg) | ||
|- | |- | ||
| | |72C, 5min | ||
|- | |- | ||
|} | |} | ||
== | == Quartz-Seq Library Preparation == | ||
'''RT Primer Removal''' | |||
''' | |||
Line 496: | Line 568: | ||
|Purified cDNA||||||||||3ul | |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 | |||
|Total|||||||||| | |||
|- | |- | ||
|} | |} | ||
''Note: can add directly to beads and transfer to new tube'' | |||
:::''Incubate @ | :::''Incubate @37C 30min'' | ||
:::''Incubate @80C 20min'' | |||
:::''Incubate @4C'' | |||
'''Poly-A Tailing''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 523: | Line 596: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |ExoI RXN||||||||||6ul | ||
|- | |||
|10x PCR Buffer||||||||||0.5ul | |||
|- | |- | ||
| | |100mM dATP||||||||||0.15ul | ||
|- | |||
|RNaseH||||||||||0.12ul | |||
|- | |- | ||
| | |TdT enzyme||||||||||0.42ul | ||
|- | |- | ||
|dH2O|||||||||| | |dH2O||||||||||3.81ul | ||
|- | |- | ||
|Total|||||||||| | |Total||||||||||11ul | ||
|- | |- | ||
|} | |} | ||
''' | :::''Incubate @37C 50 sec'' | ||
:::''Incubate @65C 10min'' | |||
:::''Incubate @4C'' | |||
'''Second Strand Synthesis''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 554: | Line 626: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |Poly-A Tailed Sample||||||||||11ul | ||
|- | |- | ||
| | |2x Terra Direct PCR Buffer||||||||||25ul | ||
|- | |- | ||
| | |10uM Tagging Primer||||||||||0.32ul | ||
|- | |- | ||
| | |Terra Polymerase||||||||||2ul | ||
|- | |- | ||
| | |dH2O||||||||||18.68ul | ||
|- | |- | ||
|Total||||||||||57ul | |||
|Total|||||||||| | |||
|- | |- | ||
|} | |} | ||
:::''Incubate @ | :::''Incubate @98C 2min 10 sec'' | ||
:::''Incubate @ | :::''Incubate @40C 1min'' | ||
:::''Incubate @68C 5min'' | |||
:::''Transfer to ice'' | |||
'''PCR Amplification''' | |||
''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 586: | Line 652: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |2nd Strand Sample||||||||||57ul | ||
|- | |- | ||
| | |2x Terra Direct PCR Buffer||||||||||25ul | ||
|- | |- | ||
| | |100uM PCR Primer||||||||||1ul | ||
|- | |- | ||
| | |dH2O||||||||||24ul | ||
|- | |- | ||
|Total|||||||||| | |Total||||||||||107ul | ||
|- | |- | ||
|} | |} | ||
::{| {{table}} | ::{| {{table}} | ||
|- | |- | ||
||||||||||| | |68C 1 sec|||||||||| | ||
|- | |- | ||
| | |98C 10s -> 65C 15s -> 68C 5min||||||||||15x (1ng); 18x (100pg); 21x (10pg) | ||
|- | |- | ||
| | |68C, 5min | ||
|- | |- | ||
|} | |} | ||
:::''Incubate | == MARS-Seq Library Preparation == | ||
'''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:'' | ||
: | ::#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 | ||
: | |||
''' | '''Second Strand Synthesis''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 644: | Line 699: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |cDNA||||||||||17ul | ||
|- | |||
|10x NEB second strand synthesis buffer||||||||||2ul | |||
|- | |||
|Second strand synthesis enzyme||||||||||1ul | |||
|- | |- | ||
| | |Total||||||||||20ul | ||
|- | |- | ||
|} | |} | ||
:::''Incubate @ | |||
::: | :::''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 657: | Line 727: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |10x Reaction Buffer (T7 high yield NEB)||||||||||2ul | ||
|- | |||
|UTP||||||||||2ul | |||
|- | |||
|ATP||||||||||2ul | |||
|- | |||
|CTP||||||||||2ul | |||
|- | |||
|GTP||||||||||2ul | |||
|- | |- | ||
| | |T7 polymerase||||||||||2ul | ||
|- | |- | ||
| | |dH2O||||||||||8ul | ||
|- | |- | ||
| | |Total||||||||||20ul | ||
|- | |- | ||
|} | |} | ||
:::''Incubate | :::''Incubate 16 hours (overnight) @ 37C''' | ||
:::'' | :::''Add 1ul Turbo DNase I and incubate @ 37C 15min | ||
::: | |||
::: | |||
:: | :''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 | |||
''' | '''Adaptor Ligation''' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 682: | Line 765: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |aRNA||||||||||3ul | ||
|- | |- | ||
| | |100uM Lig_NNNX4_idx||||||||||1ul | ||
|- | |- | ||
|} | |} | ||
:::''Incubate @70C | :::''Incubate @ 70C for 3min'' | ||
::{| {{table}} | ::{| {{table}} | ||
Line 695: | Line 778: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |aRNA/adaptor||||||||||4ul | ||
|- | |||
|10x T4 Ligase buffer||||||||||1ul | |||
|- | |- | ||
| | |DMSO||||||||||0.95ul | ||
|- | |- | ||
|10mM | |10mM ATP||||||||||1ul | ||
|- | |- | ||
| | |PEG8000||||||||||2ul | ||
|- | |- | ||
| | |T4 RNA Ligase I(10U/ul)||||||||||1ul | ||
|- | |- | ||
| | |dH2O||||||||||0ul | ||
|- | |- | ||
|Total||||||||||10ul | |Total||||||||||10ul | ||
Line 711: | Line 796: | ||
|} | |} | ||
:::''Incubate at 22C for 2hrs'' | |||
'''Reverse Transcription''' | |||
''' | |||
::{| {{table}} | ::{| {{table}} | ||
Line 723: | Line 805: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |Ligated RNA||||||||||8ul | ||
|- | |- | ||
| | |10uM MARS_RT_2||||||||||1ul | ||
|- | |- | ||
| | |10mM dNTP||||||||||1ul | ||
|- | |- | ||
|Total||||||||||10ul | |||
|Total|||||||||| | |||
|- | |- | ||
|} | |} | ||
:: | :::''Incubate @ 65C 5min'' | ||
:::''Incubate on ice 1min'' | |||
''' | |||
Line 760: | Line 824: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |RNA/Primer||||||||||10ul | ||
|- | |- | ||
| | |10x RT Buffer||||||||||2ul | ||
|- | |- | ||
| | |25mM MgCl2||||||||||0.8ul | ||
|- | |- | ||
| | |100mM DTT||||||||||2ul | ||
|- | |- | ||
|dH2O|||||||||| | |RNase Inhibitor (40U/ul)||||||||||1ul | ||
|- | |||
|SSIII (200U/ul)||||||||||1ul | |||
|- | |||
|dH2O||||||||||3.2ul | |||
|- | |- | ||
|Total||||||||||10ul | |Total||||||||||10ul | ||
Line 774: | Line 842: | ||
|} | |} | ||
:::''Incubate @ | |||
:::''Incubate @ | :::''Incubate @42C 10min'' | ||
:::''Incubate @ | :::''Incubate @50C 50min'' | ||
::: | :::''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 786: | Line 863: | ||
|||||||||||<u>Volume</u> | |||||||||||<u>Volume</u> | ||
|- | |- | ||
| | |cDNA||||||||||10ul | ||
|- | |- | ||
| | |SybrGreen KAPA 2x MM||||||||||25ul | ||
|- | |- | ||
| | |10uM P5_Rd1_PCR_F||||||||||2.5ul | ||
|- | |- | ||
| | |10uM P7_Rd2_PCR_R||||||||||2.5ul | ||
|- | |- | ||
| | |dH2O|||||||||| | ||
|- | |- | ||
|} | |} | ||
Line 803: | Line 878: | ||
::{| {{table}} | ::{| {{table}} | ||
|- | |- | ||
| | |95C 30 sec|||||||||| | ||
|- | |- | ||
| | |95C 15s -> 60C 20s -> 72C 30s||||||||||12x | ||
|- | |- | ||
|72C, 10min | |||
|72C, | |||
|- | |- | ||
|} | |} | ||
== 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]
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
- 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 ~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
- 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 ~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:
- 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
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
- 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:
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:
- 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
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
- 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
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