Blue:RNA-Seq Experiments:totoRNAseq optimizations

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Revision as of 23:08, 19 February 2014 by >B1lake (→‎RNA-Seq Library Information)
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Experimental Overview

File:TotoRNAseq expanded optimization2.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 T30VN_id1 N2.id1-2 totoRNAseq/STRT-Seq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1 N2.id3-4 totoRNAseq/STRT-Seq
3x10pg UHRR 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1 N2.id5-7 totoRNAseq/STRT-Seq
NTC 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1 N2.id8 totoRNAseq/STRT-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id9-10 totoRNAseq/STRT-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id11-12 totoRNAseq/STRT-Seq
3x10pg UHRR 0.05U RNase3 Frag/Affymetrix PAP 4 min TSO.r06 T30VN_id1 N2.id13-15 totoRNAseq/STRT-Seq
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id16 totoRNAseq/STRT-Seq
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
3x10pg UHRR 2min Mg Frag/Enzymatics PAP 5 min Lig_NNNX4_id05-7 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
NTC 2min Mg Frag/Enzymatics PAP 5 min Lig_NNNX4_id08 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_id03-4 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
3x10pg UHRR 0.05U RNase3 Frag/Affymetrix PAP 5 min Lig_NNNX4_id05-7 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min Lig_NNNX4_id08 MARS_T20N_id01 n/a totoRNAseq/MARS-Seq
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN N2.id17-18 totoRNAseq/Nugen
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN N2.id19-20 totoRNAseq/Nugen
3x10pg UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN.id1 N2.id21-23 totoRNAseq/Nugen
NTC 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN_id1 N2.id24 totoRNAseq/Nugen
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_N8 T30VN_id1 N2.id225-26 totoRNAseq/Nugen
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_N8 T30VN_id1 N2.id27-28 totoRNAseq/Nugen
3x10pg UHRR 0.05U RNase3 Frag/Affymetrix PAP 4 min STRT_N8 T30VN_id1 N2.id29-31 totoRNAseq/Nugen
NTC 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_N8 T30VN_id1 N2.id32 totoRNAseq/Nugen


Primer sequences [1]


totoRNAseq Protocol Schemes

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
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


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

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

STRT-Seq 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 10min
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)
72C, 5min


  • Run 5ul on gel:



Results:

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


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


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


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


PCR Amplification

Volume
2nd Strand Sample 57ul
2x Terra Direct PCR Buffer 25ul
100uM Tagging 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


  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
  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

Nugen Ovation Library Preparation

Second Strand Synthesis


Volume
cDNA 3ul
2x PhusionHF Flex 5ul
10uM STRT_N8 0.3ul
RNaseH 0.1ul
dH2O 1.6ul
Total 10ul
Incubate @37C 20min
Incubate @98C 30sec
Incubate @25C 30min
Incubate @70C 10min


PCR Amplification

Volume
2nd Strand Sample 10ul
2x PhusionHF Flex 50ul
100uM P1-STRT 1ul
100uM PCR_R_N2.id 1ul
dH2O 38ul
Total 100ul


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

Nextera XT for Quartz-Seq