Blue:RNA-Seq Experiments:03052014: Difference between revisions

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::*Check library size by gel (ran 5ul on UREA gel):
::*Check library size by gel (ran 5ul on UREA gel):


[[File:03052014_PCR2_Gel1_STRT.jpg|75px]]
[[File:03052014_PCR2_Gel1_STRT.jpg|100px]]


::Ran PCR using 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]
::[http://genome-tech.ucsd.edu/LabNotes/index.php/Blue:Data/2014-5-12 Final Libraries Ran on Gel]

Revision as of 01:09, 19 June 2014

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 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 [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
  1. Add 25ul 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 dH20; incubate 2min RT; transfer to new tube and vacuum spin to ~3ul (~8min)

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
  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

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.


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

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
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...


Repeat Experiment

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
IS PCR Primer (10uM) 2ul
H2O 13ul
Total 50ul


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


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


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


Ran 5ul onto gel:

File:03052014 PCR1 Gel1 Quartz.jpg

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:
  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 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


  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


Beads Purification:
  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

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
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

Volume
Ligated RNA 4ul
10uM MARS_RT_2 0.5ul
10mM dNTP 0.5ul
Total 5ul


Incubate @ 65C 5min
Incubate on ice 1min


Volume
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

  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 10ul


95C 30 sec
95C 15s -> 60C 20s -> 72C 30s up to 20x
72C, 10min


File:03052014 MARS PCR1.jpg

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:
Sample Condition [cDNA] ng/ul Final Library Volume to Combine N2 Barcode NexteraXT Volume for 0.1-0.2ng/ul Volume dH2O Nextera Barcode Sten Tagmentation Volume for 1ng/ul Volume dH20 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

Volume
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

Volume
Tagmented Sample 12.5ul
Nextera PCR Mix 7.5ul
S5xx Primer 2.5ul
S7xx Primer 2.5ul
Total 20ul


Mix and centrifuge


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

  • 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


Volume
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

Ran PCR using and ran 5ul on gel:

File:03052014 PCR2 Gel2 STRT.jpg

Final Libraries Ran on Gel