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 optimization2.jpg|600px]]


== Purpose ==
== Purpose ==

Revision as of 19:59, 13 February 2014

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
  • Comparison of Enzymatics and Affymetrix PAP

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
2xNTC 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1 N2.id5-6 totoRNAseq/STRT-Seq
10x10pg UHRR/2xNTC 2min Mg Frag/Enzymatics PAP 5 min TSO.r06 T30VN_id1-12 N2.id7 totoRNAseq/STRT-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id8-9 totoRNAseq/STRT-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id10-11 totoRNAseq/STRT-Seq
2xNTC 0.1U RNase3 Frag/Affymetrix PAP 10 min TSO.r06 T30VN_id1 N2.id12-13 totoRNAseq/STRT-Seq
10x10pg UHRR/2xNTC 0.05U RNase3 Frag/Affymetrix PAP 4 min TSO.r06 T30VN_id1-12 N2.id14 totoRNAseq/STRT-Seq
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_T24 T30VN_id1 N2.id15-16 totoRNAseq/Quartz-Seq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_T24 T30VN_id1 N2.id17-18 totoRNAseq/Quartz-Seq
2xNTC 2min Mg Frag/Enzymatics PAP 5 min STRT_T24 T30VN_id1 N2.id19-20 totoRNAseq/Quartz-Seq
10x10pg UHRR/2xNTC 2min Mg Frag/Enzymatics PAP 5 min STRT_T24 T30VN_id1-12 N2.id21 totoRNAseq/Quartz-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_T24 T30VN_id1 N2.id22-23 totoRNAseq/Quartz-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_T24 T30VN_id1 N2.id24-25 totoRNAseq/Quartz-Seq
2xNTC 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_T24 T30VN_id1 N2.id26-27 totoRNAseq/Quartz-Seq
10x10pg UHRR/2xNTC 0.05U RNase3 Frag/Affymetrix PAP 5 min STRT_T24 T30VN_id1-12 N2.id28 totoRNAseq/Quartz-Seq
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_RA3 T30VN_id1/RL_Add.T7 N2.id29-30 totoRNAseq/Cel-Seq
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_RA3 T30VN_id1/RL_Add.T7 N2.id31-32 totoRNAseq/Cel-Seq
2xNTC 2min Mg Frag/Enzymatics PAP 5 min STRT_RA3 T30VN_id1/RL_Add.T7 N2.id33-34 totoRNAseq/Cel-Seq
10x10pg UHRR/2xNTC 2min Mg Frag/Enzymatics PAP 5 min STRT_RA3 T30VN_id1-12/RL_Add.T7 N2.id35 totoRNAseq/Cel-Seq
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_RA3 T30VN_id1/RL_Add.T7 N2.id36-37 totoRNAseq/Cel-Seq
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_RA3 T30VN_id1/RL_Add.T7 N2.id38-39 totoRNAseq/Cel-Seq
2xNTC 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_RA3 T30VN_id1/RL_Add.T7 N2.id40-41 totoRNAseq/Cel-Seq
10x10pg UHRR/2xNTC 0.05U RNase3 Frag/Affymetrix PAP 4 min STRT_RA3 T30VN_id1-12/RL_Add.T7 N2.id42 totoRNAseq/Cel-Seq
2x1ng UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN_id1 N2.id43-44 totoRNAseq/Nugen
2x100pg UHRR 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN_id1 N2.id45-46 totoRNAseq/Nugen
2xNTC 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN_id1 N2.id47-48 totoRNAseq/Nugen
10x10pg UHRR/2xNTC 2min Mg Frag/Enzymatics PAP 5 min STRT_N8 T30VN_id1-12 N2.id49 totoRNAseq/Nugen
2x1ng UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_N8 T30VN_id1 N2.id50-51 totoRNAseq/Nugen
2x100pg UHRR 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_N8 T30VN_id1 N2.id52-53 totoRNAseq/Nugen
2xNTC 0.1U RNase3 Frag/Affymetrix PAP 10 min STRT_N8 T30VN_id1 N2.id54-55 totoRNAseq/Nugen
10x10pg UHRR/2xNTC 0.05U RNase3 Frag/Affymetrix PAP 4 min STRT_N8 T30VN_id1-12 N2.id56 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
0.5U/ul Enzymatics PAP (1:10d in H2O) 0.5ul + 0.3ul dH2O
or 600U/ul Affymetrix PAP 0.2ul + 0.6ul dH2O
40U/ul Rnase Inhibitor 0.2ul
Total 4ul


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


Beads Purification
  1. Add 15ul beads per well
  2. Allow to bind 15 min, combine wells (10pg only)
  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 (10ul for 10x10pg), 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
or 600U/ul Affymetrix PAP 0.2ul + 0.3ul dH2O


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


Incubate @ 42C 30 min


Beads Purification
  1. Add 15ul beads per well
  2. Allow to bind 15 min, combine wells (10pg only)
  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 (10ul for 10x10pg), 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 PhusionHF Flex 25ul
10uM STRT_T24 0.32ul
dH2O 20.68ul
Total 57ul
Incubate @98C 30sec
Incubate @40C 1min
Incubate @68C 5min


PCR Amplification

Volume
2nd Strand Sample 57ul
2x PhusionHF Flex 25ul
100uM P1-STRT 1ul
100uM PCR_R_N2.id 1ul
dH2O 23ul
Total 107ul


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

Cel-Seq Library Preparation

Second Strand Synthesis


Volume
Purified cDNA 3ul
FS Buffer 0.2ul
SS Buffer 1ul
10mM dNTP 0.8ul
DNA Polymerase 0.2ul
RNaseH 0.1ul
dH2O 4.7ul
Total 10ul


Incubate @16C 2 hours


T7 Primer Addition


Volume
Second Strand Sample 10ul
KAPA qPCR MM 25ul
RL.Add.T7 Primer (10uM) 0.5ul
dH2O 14.5ul
Total 50ul
Incubate @94C 2min
Incubate @60C 2min
Incubate @72C 5min

Beads Purification

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


IVT

Volume
Purified T7-containing Sample 6.4ul
ATP 1.6ul
GTP 1.6ul
CTP 1.6ul
UTP 1.6ul
10x T7 buffer 1.6ul
T7 Enzyme 1.6ul
Total 16ul
Incubate @37C (lid 70C) 13 hours
Incubate @4C

Quantification of aRNA

  • Determine concentration of amplified RNA using Qubit
  • Dilute 5-10ng RNA into 16ul dH2O

Phosphatase Treatment

Volume
aRNA 16ul
10x Phosphatase buffer 2ul
Anarctic phosphatase 1ul
RNase Inhibitor 1ul
Total 20ul
Incubate @37C 30min
Incubate @65C 5min
Incubate @4C Indefinitely

PNK Treatment

Volume
Phos Treated aRNA 20ul
dH2O 17ul
10x phosphatase buffer 5ul
ATP (10mM, from Illumina kit) 5ul
RNase Inhibitor 1ul
PNK 2ul
Total 50ul
Incubate @37C 60min

RNA Cleanup: (Zymo RNA Clean and Concentrator-5)

  • 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


3'Adaptor Ligation

Volume
Phos/PNK aRNA 5ul
3' STRT_RA3 (1:5 dilution?) 1ul
Incubate @70C 2min
transfer to ice
Volume
aRNA/adaptor 6ul
5x HM Ligation Buffer (HML, Illumina kit) 2ul
RNase Inhibitor 1ul
T4 RNA Ligase 2, truncated 1ul
Incubate @28C 1 hour (unheated lid)
Still in thermocycler: add 1ul stop solution (STP)
Pipette entire volume 6-8 times to mix
Incubate @28C 15min
Transfer to ice
Add 3ul dH2O


Reverse Transcription

Volume
Adaptor-ligated RNA 6ul (store remaining at -80C)
RNA RT Primer (STRT_RTP) 1ul
Incubate @70C 2min
Immediately transfer to ice
Volume
RNA/primer 7ul
10x RT Buffer 1ul
10mM dNTP 0.5ul
100mM DTT 0.5ul
RNase Inhibitor 0.5ul
SuperScriptIII 0.5ul
Total 10ul


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


PCR Amplification

Volume
RT Reaction 10ul
2x PhusionHF Flex 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 (10ng)
72C, 5min

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