Blue:RNA-Seq Experiments:03052014
Experimental Overview
File:TotoRNAseq expanded optimization3.jpg
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
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 | 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 | 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 [1]
cDNA Preparation: RNAseIII-Based Fragmentation
- 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 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
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 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
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 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
cDNA Preparation: Mg-based Fragmentation
- 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 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
Volume 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
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:
File:03052014 PCR1 Gel1 toto.jpg
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.
UMI-STRT-Seq Library Preparation
- 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 10uM 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 7ul on gel:
File:03052014 PCR1 Gel1 STRT.jpg
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
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 @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
File:03052014 PCR1 Gel1 Smart2.jpg
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...
Quartz-Seq Library Preparation
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
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
Volume 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:
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
Quantification:
- Removed 4ul for RT (see below)
- Added 14ul to 6ul ligated RNA
- Beads purified (1.2x)
- Quantified by Qubit:
Invitrogen ' ' ' ' ' ' Qubit(TM) Fluorometer 3/27/2014 2:26:11 PM Quant-iT RNA Sample Concentration in the Qubit µL used Dilution Sample Concentration 1 79.3 ng/mL 1 200.0 15.9 µg/mL 2 115 ng/mL 1 200.0 23.0 µg/mL 3 46.1 ng/mL 1 200.0 9.21 µg/mL 4 46.5 ng/mL 1 200.0 9.30 µg/mL 5 Out Of Range (too low) 6 102 ng/mL 1 200.0 20.4 µg/mL
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