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Blue:RNA-Seq Experiments:03052014
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== Experimental Overview == [[File:TotoRNAseq expanded optimization3.jpg|600px]] == Purpose == *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 == {| {{table}} | align="center" style="background:#f0f0f0;"|'''Library ID''' | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Condition''' | align="center" style="background:#f0f0f0;"|'''5' Adaptor''' | align="center" style="background:#f0f0f0;"|'''3' Adaptor''' | align="center" style="background:#f0f0f0;"|'''N2 PCR Rev Primer''' | align="center" style="background:#f0f0f0;"|'''RNA-Seq Protocol''' |- | BL_totoRNAseq_1ngUHRR_||2x1ng UHRR||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T20VN_id1||N2.id1-2||totoRNAseq |- | ||2x100pg UHRR||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T20VN_id1||N2.id3-4||totoRNAseq |- | ||10x10pg UHRR (combined)||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T20VN_id1-10||N2.id5||totoRNAseq |- | ||NTC||2min Mg Frag/Enzymatics PAP 5 min||TSO.r06||T20VN_id11||N2.id6||totoRNAseq |- | ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||TSO.r06||T20VN_id1||N2.id7-8||totoRNAseq |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||TSO.r06||T20VN_id1||N2.id9-10||totoRNAseq |- | ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 4 min||TSO.r06||T20VN_id1-10||N2.id11||totoRNAseq |- | ||NTC||0.05U RNase3 Frag/Affymetrix PAP 4 min ||TSO.r06||T20VN_id11||N2.id12||totoRNAseq |- | ||2x1ng UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||T20VN_id11||N2.id13-14||totoRNAseq |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||C1-P1-RNA-TSO||T20VN_id11||N2.id15-16||totoRNAseq |- | ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 4 min||C1-P1-RNA-TSO||T20VN_id1-10||N2.id17||totoRNAseq |- | ||NTC||0.05U RNase3 Frag/Affymetrix PAP 4 min ||C1-P1-RNA-TSO||T20VN_id11||N2.id18||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 4 min||C1-P1-RNA-TSO||C1-P1-T31||C1-TN5-x||totoRNAseq/UMI-STRT-Seq |- | ||NTC||0.05U RNase3 Frag/Affymetrix PAP 4 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||0.05U RNase3 Frag/Affymetrix PAP 4 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||0.05U RNase3 Frag/Affymetrix PAP 4 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_id01-2||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- | ||2x100pg UHRR||0.1U RNase3 Frag/Affymetrix PAP 10 min||Lig_NNNX4_id3-4||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- | ||10x10pg UHRR (combined)||0.05U RNase3 Frag/Affymetrix PAP 4 min||Lig_NNNX4_id5||MARS_T20N_id01-10||n/a||totoRNAseq/MARS-Seq |- | ||NTC||0.05U RNase3 Frag/Affymetrix PAP 4 min||Lig_NNNX4_id6||MARS_T20N_id01||n/a||totoRNAseq/MARS-Seq |- |} Primer sequences [http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:RNAseq_Primer_List#totoRNA_5.27_primer_sequence] == cDNA Preparation: RNAseIII-Based Fragmentation == :'''RNA''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2ng/ul or 0.2ng/ul or 0.02ng/ul ||||||||||0.5ul |- |1:10E4 or 1:10E5 or 1:10E6 ERCC ||||||||||0.2ul |- |xuM PolyT primer (see below)||||||||||0.1ul |- |dH2O||||||||||0.2ul |- |Total||||||||||1ul |- |} Note: NTC with 1:10E6 ERCC and no RNA - treated as 10pg... :::''Incubate @ 72C 3 min'' :::''Incubate @ 37C 1 min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |10x RNaseIII buffer||||||||||0.2ul |- |RNase III||||||||||0.1ul |- |dH20||||||||||0.7ul |- |Total||||||||||2ul |- |} :::''Incubate @ 37C 1 min'' :'''3' Tailing''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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 T20VN.id [totoRNAseq]'' :::''Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]'' :::''Add 1ul of 10uM Smart2-T30VN [Smart-seq2]'' :::''Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]'' :::''Add 1ul of 2uM MARS_T20N_id [MARS-Seq]'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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 60 min'' :::''For MARS-seq - continue to that section'' :'''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 dH20; incubate 2min RT; transfer to new tube and vacuum spin to ~3ul (~8min) == cDNA Preparation: Mg-based Fragmentation == :'''RNA FRAGMENTATION''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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 T20VN.id [totoRNAseq]'' :::''Add 1ul of 12uM C1-P1-T31 [UMI-STRT-Seq]'' :::''Add 1ul of 10uM Smart2-T30VN [Smart-seq2]'' :::''Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]'' :::''Add 1ul of 2uM MARS_T20N_id [MARS-Seq]'' :'''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |PAP RNA ||||||||||5.1ul |- |5xRT Buffer||||||||||1ul |- |10mM dNTP Mix||||||||||1ul |- |100mM DTT||||||||||0.25ul |- |Betaine (5M)||||||||||2ul |- |Smarter MMLV (200U/ul)||||||||||0.25ul |- |dH2O||||||||||0.4ul |- |Total||||||||||10ul |- |} :::''Incubate @ 42C 60 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 == :'''TSO Reaction''' ::{| {{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 20min'' :::''Incubate @70C 10min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |10uM P1-STRT||||||||||1ul |- |10uM PCR_R_N2_id||||||||||1ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 72C 20s||||||||||9x (1ng); 12x (100pg) |- |72C, 5min |- |} *Run 5ul on gel: [[File:03052014_PCR1_Gel1_toto.jpg|300px]] Results: Decent amplification for all conditions. Negative control (NC) which does have ERCC (10pg level) shows a distinct band at around 300bp for both Mg protocol (above) and the RNaseIII protocol (see STRT gel below). Mg protocol does show more high molecular weight fragments. * Beads purified (0.8x) twice and eluted in 10ul. '''Repeat RNaseIII Protocol using Sten TSO''' :*Repeated exactly the same way except with the following substitutions: :**TSO.r06 replaced with C1-P1-RNA-TSO :**P1-STRT replaced with C1-P1-PCR-2 :*Run 5ul on gel: [[File:03052014_PCR1_Gel1_toto_sten.jpg|200px]] Note: Possible mistake with the quantification of the input RNA obtained from Illumina - may be double that intended. Indicates that the efficiency of this TSO in the totoRNAseq protocol might be too low to be truly viable... == UMI-STRT-Seq Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||7ul |- |10x Advantage 2 Buffer||||||||||7ul |- |10uM 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 7ul on gel: [[File:03052014_PCR1_Gel1_STRT.jpg|600px]] Results: Mg protocol does not seem to be compatible with Sten's latest STRT protocol (using RNA TSO) - possible carry over of Enzymatics PAP through beads purification. RNaseIII protocol (Affymetrics PAP) seems fine for all RNA amounts, but does show a ~600bp band. The NC for the RNaseIII STRT protocol does seem clean. Amplification levels for the RNaseIII protocol are lower than the totoRNAseq protocol. *Beads Purified (0.9x) twice and eluted in 10ul. Quantification using Qubit: == Smart-Seq2 Library Preparation == :'''TSO Reaction''' ::{| {{table}} |- |||||||||||<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||||||||||0.59ul |- |Total||||||||||10ul |- |} :::''Incubate @42C 20min'' :::''Incubate @70C 10min'' :::''Incubate @10C hold'' :'''PCR (KAPA-HiFi)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x KAPA HiFi Hotstart ReadyMix||||||||||12.5ul |- |IS PCR Primer (10uM)||||||||||0.25ul |- |H2O||||||||||2.25ul |- |Total||||||||||25ul |- |} ::{| {{table}} |- |98C 3min|||||||||| |- |98C 20s -> 67C 15s -> 72C 1min||||||||||15x (1ng); 18x (100pg); 21x (10pg) |- |72C, 5min |- |} [[File:03052014_PCR1_Gel1_Smart2.jpg|400px]] Results: Can see that the PCR did not work. Maybe the conditions are sub-optimal for fragmented RNA. Could try similar conditions as the STRT-seq or totoRNAseq... '''Repeat Experiment''' :'''TSO Reaction''' ::{| {{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 20min'' :::''Incubate @70C 10min'' :'''PCR (PhusionHF)''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |TSO Reaction||||||||||10ul |- |2x Buffer||||||||||25ul |- |IS PCR Primer (10uM)||||||||||2ul |- |H2O||||||||||13ul |- |Total||||||||||50ul |- |} ::{| {{table}} |- |98C 30 sec|||||||||| |- |98C 15s -> 58C 20s -> 72C 30s||||||||||6x |- |98C 10s -> 64C 20s -> 72C 20s||||||||||9x (1ng); 12x (100pg); 15x (10pg) |- |72C, 5min |- |} *Run 5ul on gel: [[File:03052014_PCR1_Gel1_Smart2_2.jpg|400px]] Results: Appears that the Smart-seq2 protocol is also incompatible with the Mg RNA fragmentation protocol (likely due to the enzymatics PAP). For RNaseIII fragmented RNA, the efficiency looks to be too low to amplify 10pg libraries... therefore the Smart-seq2 adaptation may not work well enough to continue. cDNA quantification is needed to determine which samples can be used for Nextera library preparation. == Quartz-Seq Library Preparation == '''RT Primer Removal''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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''' ::{| {{table}} |- |||||||||||<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 |- |} :::''Incubate @98C 2min 10 sec'' :::''Incubate @40C 1min'' :::''Incubate @68C 5min'' :::''Transfer to ice'' '''PCR Amplification''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |2nd Strand Sample||||||||||57ul |- |2x Terra Direct PCR Buffer||||||||||25ul |- |100uM PCR Primer||||||||||1ul |- |dH2O||||||||||24ul |- |Total||||||||||107ul |- |} ::{| {{table}} |- |68C 1 sec|||||||||| |- |98C 10s -> 65C 15s -> 68C 5min||||||||||15x (1ng); 18x (100pg); 21x (10pg) |- |68C, 5min |- |} Ran 5ul onto gel: [[File:03052014_PCR1_Gel1_Quartz.jpg|500px]] Results: Mg protocol also is problematic for Quartz-seq. I'm not sure why this is the case since no RNA adaptor primer was used... Looks like the RNaseIII protocol did work down to the 10pg level, though quantification is needed to determine whether there is enough yield for Nextera library preparation. Note that while the yields look lower, less sample was run on the gel compared to totoRNAseq (5ul of 107ul vs 5ul of 50ul). == 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 dH2O, incubate 2min, transfer to new tube (Note: stored overnight at -20C at this point) '''Second Strand Synthesis''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |dH20||||||||||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: ::{| {{table}} |- |||||||||||<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 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}} |- |||||||||||<u>Volume</u> |- |aRNA||||||||||3ul |- |100uM Lig_NNNX4_idx||||||||||1ul |- |} :::''Incubate @ 70C for 3min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |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'' Quantification: :*Removed 4ul for RT (see below) :*Added 14ul to 6ul ligated RNA :*Beads purified (1.2x) :*Quantified by Qubit: ::{| {{table}} | align="center" style="background:#f0f0f0;"|'''Invitrogen''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''''' |- | Qubit(TM) Fluorometer|||||||||||| |- | 3/27/2014 2:26:11 PM|||||||||||| |- | |||||||||||| |- | Quant-iT RNA|||||||||||| |- | Sample||Concentration in the Qubit||||µL used||Dilution||Sample Concentration|| |- | RIII_id1||79.3||ng/mL||1||200.0||15.9||µg/mL |- | RIII_id2||115||ng/mL||1||200.0||23.0||µg/mL |- | RIII_id3||46.1||ng/mL||1||200.0||9.21||µg/mL |- | RIII_id4||46.5||ng/mL||1||200.0||9.30||µg/mL |- | RIII_id5||Out Of Range (too low)|||||||||| |- | RIII_id6 (NTC)||102||ng/mL||1||200.0||20.4||µg/mL |} '''Reverse Transcription''' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Ligated RNA||||||||||4ul |- |10uM MARS_RT_2||||||||||0.5ul |- |10mM dNTP||||||||||0.5ul |- |Total||||||||||5ul |- |} :::''Incubate @ 65C 5min'' :::''Incubate on ice 1min'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |RNA/Primer||||||||||5ul |- |10x RT Buffer||||||||||1ul |- |25mM MgCl2||||||||||0.4ul |- |100mM DTT||||||||||1ul |- |RNase Inhibitor (40U/ul)||||||||||0.5ul |- |SSIII (200U/ul)||||||||||0.5ul |- |dH2O||||||||||1.6ul |- |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''' ::{| {{table}} |- |||||||||||<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||||||||||10ul |- |} ::{| {{table}} |- |95C 30 sec|||||||||| |- |95C 15s -> 60C 20s -> 72C 30s||||||||||up to 20x |- |72C, 10min |- |} [[File:03052014_MARS_PCR1.jpg|400px]] Results: Amplification pattern implies problem with protocol... needs further optimization... == Final Library Preparation == ''cDNA Quantification'' :'''Quantification of 1ul out of 10ul Beads purified libraries using Qubit:''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Condition''' | align="center" style="background:#f0f0f0;"|'''[cDNA] ng/ul''' | align="center" style="background:#f0f0f0;"|'''Final Library''' | align="center" style="background:#f0f0f0;"|'''Volume to Combine''' | align="center" style="background:#f0f0f0;"|'''N2 Barcode''' | align="center" style="background:#f0f0f0;"|'''NexteraXT''' | align="center" style="background:#f0f0f0;"|'''Volume for 0.1-0.2ng/ul''' | align="center" style="background:#f0f0f0;"|'''Volume dH2O''' | align="center" style="background:#f0f0f0;"|'''Nextera Barcode''' | align="center" style="background:#f0f0f0;"|'''Sten Tagmentation''' | align="center" style="background:#f0f0f0;"|'''Volume for 1ng/ul''' | align="center" style="background:#f0f0f0;"|'''Volume dH20''' | align="center" style="background:#f0f0f0;"|'''Sten Barcode''' |- | 1||TotoRNAseq Mg 1ng||8.95||Yes||2||1||N/A||||||||N/A|||||| |- | 2||TotoRNAseq Mg 1ng||8.84||Yes||2.02||2||N/A||||||||N/A|||||| |- | 3||TotoRNAseq Mg 100pg||8.57||Yes||2.09||3||N/A||||||||N/A|||||| |- | 4||TotoRNAseq Mg 100pg||10||Yes||1.79||4||N/A||||||||N/A|||||| |- | 5||TotoRNAseq Mg 10x10pg||15.2||Yes||N/A||5||N/A||||||||N/A|||||| |- | 6||TotoRNAseq Mg NTC||4.04||Yes||2||6||N/A||||||||N/A|||||| |- | 7||TotoRNAseq RIII 1ng||3.13||Yes||1||7||N/A||||||||N/A|||||| |- | 8||TotoRNAseq RIII 1ng||2.96||Yes||1.06||8||N/A||||||||N/A|||||| |- | 9||TotoRNAseq RIII 100pg||1.38||Yes||2.27||9||N/A||||||||N/A|||||| |- | 10||TotoRNAseq RIII 100pg||0.977||Yes||3.20||10||N/A||||||||N/A|||||| |- | 11||TotoRNAseq RIII 10x10pg||1.39||Yes||N/A||11||N/A||||||||N/A|||||| |- | 12||TotoRNAseq RIII NTC||-||Yes||2||12||N/A||||||||N/A|||||| |- | 13||TotoRNAseq RIII StenTSO 1ng||1.21||Yes||1||13||N/A||||||||N/A|||||| |- | 14||TotoRNAseq RIII StenTSO 1ng||1.54||Yes||0.785714286||14||N/A||||||||N/A|||||| |- | 15||TotoRNAseq RIII StenTSO 100pg||0.948||Yes||1.276371308||15||N/A||||||||N/A|||||| |- | 16||TotoRNAseq RIII StenTSO 100pg||0.844||Yes||1.433649289||16||N/A||||||||N/A|||||| |- | 17||TotoRNAseq RIII StenTSO 10x10pg||0.19||Yes||6.368421053||17||N/A||||||||N/A|||||| |- | 18||TotoRNAseq RIII StenTSO NTC||-||Yes||1||18||N/A||||||||N/A|||||| |- | 19||STRT RIII 1ng||3.54||No||||||N/A||||||||Yes||1.694915254||4.305084746||C1-TN5-1 |- | 20||STRT RIII 1ng||3.35||No||||||N/A||||||||Yes||1.791044776||4.208955224||C1-TN5-2 |- | 21||STRT RIII 100pg||2.78||No||||||N/A||||||||Yes||2.158273381||3.841726619||C1-TN5-3 |- | 22||STRT RIII 100pg||2.99||No||||||N/A||||||||Yes||2.006688963||3.993311037||C1-TN5-4 |- | 23||STRT RIII 10pg||2.42||No||||||N/A||||||||Yes||2.479338843||3.520661157||C1-TN5-5 |- | 24||STRT RIII 10pg||2.22||No||||||N/A||||||||Yes||2.702702703||3.297297297||C1-TN5-6 |- | 25||STRT RIII 10pg||2.86||No||||||N/A||||||||Yes||2.097902098||3.902097902||C1-TN5-7 |- | 26||STRT RIII NTC||0.696||No||||||N/A||||||||Yes||6||0||C1-TN5-8 |- | 27||Smart2 RIII 1ng||1.48||No||||||Yes||0.68||4.32||S501/N701||N/A|||||| |- | 28||Smart2 RIII 1ng||1.47||No||||||Yes||0.68||4.32||S502/N702||N/A|||||| |- | 29||Smart2 RIII 100pg||0.52||No||||||Yes||1.92||3.08||S503/N703||N/A|||||| |- | 30||Smart2 RIII 100pg||0.49||No||||||Yes||2.04||2.96||S504/N704||N/A|||||| |- | 31||Smart2 RIII 10pg||0.13||No||||||Yes||3.85||1.15||S505/N705||N/A|||||| |- | 32||Smart2 RIII 10pg||0.12||No||||||Yes||4.17||0.83||S506/N706||N/A|||||| |- | 33||Smart2 RIII 10pg||-||No||||||Yes||5.00||0.00||S507/N707||N/A|||||| |- | 34||Smart2 RIII NTC||-||No||||||Yes||5.00||0.00||S508/N708||N/A|||||| |- | 35||Quartz RIII 1ng||13.8||No||||||Yes||0.58||39.42||S501/N709||N/A|||||| |- | 36||Quartz RIII 1ng||10.4||No||||||Yes||0.77||39.23||S502/N710||N/A|||||| |- | 37||Quartz RIII 100pg||3.49||No||||||Yes||0.86||14.14||S503/N711||N/A|||||| |- | 38||Quartz RIII 100pg||3.73||No||||||Yes||0.80||14.20||S504/N712||N/A|||||| |- | 39||Quartz RIII 10pg||1.95||No||||||Yes||1.54||13.46||S505/N701||N/A|||||| |- | 40||Quartz RIII 10pg||3.63||No||||||Yes||0.83||14.17||S506/N702||N/A|||||| |- | 41||Quartz RIII 10pg||4.93||No||||||Yes||0.61||14.39||S507/N703||N/A|||||| |- | 42||Quartz RIII NTC||1.83||No||||||Yes||1.64||13.36||S508/N704||N/A|||||| |} ''Nextera Tagmentation'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagment DNA Buffer||||||||||5ul |- |Amplification Tagment Mix||||||||||2.5ul |- |Diluted Sample||||||||||2.5ul |- |Total||||||||||10ul |- |} ::*Vortex and spin ::*Incubate 55C 5 min ::*Hold 10C ::*Once sample reaches 10C add 2.5ul of NT buffer in block ::*Vortex and spin ''Nextera Amplification'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |Tagmented Sample||||||||||12.5ul |- |Nextera PCR Mix||||||||||7.5ul |- |S5xx Primer||||||||||2.5ul |- |S7xx Primer||||||||||2.5ul |- |Total||||||||||20ul |- |} ::''Mix and centrifuge'' ::{| {{table}} |- |72C 3 min|||||||||| |- |95C 30s|||||||||| |- |95C 10s -> 55C 30s -> 72C 60s||||||||||12x |- |72C, 5min |- |} ::*Run 2.5ul on gel: [[File:03052014_PCR2_Gel1_Nextera.jpg|500px]] ::*Combined 5ul of each for Smart2 ::*Combined 5ul each of 1ng/100pg/NTC and 7.5ul each of 10pg for Quartz-seq ::*Beads purified twice (0.8x volume) and resuspended in 10ul ''Sten Tagmentation'' ::{| {{table}} |- |||||||||||<u>Volume</u> |- |5x Tagmentation Buffer||||||||||5ul |- |Transposome stock (Epicenter)||||||||||6ul |- |Sample||||||||||3ul |- |dH2O||||||||||11ul |- |Total||||||||||25ul |- |} ::*Incubate @55C for 8 min ::*Cool on ice ::*Wash 100ul Dynabeads MyOne Streptavidin C1 Beads with 2xBWT and resuspended in 2ml 2xBWT ::*Add 20ul beads to each well and incubate 5min ::*Combine beads, immobilize and discard supernatant ::*Wash beads with 100ul TNT ::*Wash beads with 100ul Qiagen Qiaquick PB ::*Wash beads twice with 100ul TNT ::*Resuspend beads in 100ul restriction mix: :::*10ul NEBuffer 4 :::*2ul PvuI-HF (0.4U/ul) :::*88ul dH2O ::*Incubate at 37C for 1 hour ::*Wash beads 3x with TNT ::*Elute in 10ul 100mM NaOH, incubate 5 min then remove to new tube ::*Neutralize by adding: :::*10ul 100mM HCl :::*5ul Neutralization buffer ::*Check library size by gel (ran 5ul on UREA gel): [[File:03052014_PCR2_Gel1_STRT.jpg|100px]] ::Ran Sybr PCR using C1_P1_PCR_2/C1_TN5_U for 12 cycles (25ul total) and ran 5ul on gel: [[File:03052014_PCR2_Gel2_STRT.jpg|100px]] ::[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:Data/2014-5-12 Final Libraries Ran on Gel]
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