Blue:RNA-Seq Experiments:06042014

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Purpose[edit]

  • Improve upon totoQuartzSeq protocol - optimize PCR Cylcles for 1ng UHRR

RNA-Seq Library Information[edit]

Library ID Sample Condition 5' Adaptor 3' Adaptor N2 PCR Rev Primer RNA-Seq Protocol
2x1ng UHRR RIII Frag - 11 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR RIII Frag - 13 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR RIII Frag - 15 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
NTC RIII Frag - 15 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR Mg Frag - 11 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR Mg Frag - 13 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
2x1ng UHRR Mg Frag - 15 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq
NTC Mg Frag - 15 PCR cycles Quartz_Tagging Quartz-RT Quartz-Suppression totoRNAseq/Quartz-Seq


Primer sequences [1]

cDNA Preparation: RNAseIII-Based Fragmentation[edit]

RNA
Volume
2ng/ul UHRR 0.5ul
1:10e4 ERCC 0.2ul
0.1uM Quartz-RT (see below) 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 Quartz-RT [Quartz-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
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 ExoI reaction (see below):

cDNA Preparation: Mg-based Fragmentation[edit]

RNA FRAGMENTATION
Volume
2ng/ul UHRR 0.5ul
1:10e4 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
40U/ul 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
600U/ul Affymetrix PAP 0.2ul
dH2O 0.2ul
Total 4


Incubate @ 37C 10 min
Transfer to Ice
Add 1ul of 0.1uM Quartz-RT [Quartz-Seq]


Reverse Transcription
Volume
PAP RNA 5ul
5xRT Buffer 1ul
10mM dNTP Mix 1ul
100mM DTT 0.25ul
Betaine (5M) 2ul
40U/ul Rnase Inhibitor 0.1ul
Smarter MMLV (200U/ul) 0.25ul
dH2O 0.4ul
Total 10ul


Incubate @ 42C 60 min
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 ExoI reaction (below):

Quartz-Seq Library Preparation[edit]

RT Primer Removal


Volume
10x PCR Buffer (homemade Takara) 0.4ul
10x Exo I buffer (epicentre) 0.2ul
100mM DTT 0.2ul
Exonuclease I (epicentre) 0.6ul
dH2O 4.6ul
Total 6ul


Takara Taq PCR Buffer:
Volume
1M Tris.HCl (pH 8.3) 100ul (100mM Final)
1M KCl 500ul (500mM Final)
1M MgCl2 15ul (15mM Final)
dH2O 385ul
Total 1ml



Add directly to beads and mix
Incubate @37C 30min
Incubate @80C 20min
Incubate @4C


Poly-A Tailing

Volume
ExoI RXN 6ul
10x PCR Buffer 0.5ul
10mM dATP 1.5ul
RNaseH (ambion) 0.12ul
TdT enzyme (Roche 03333566001) 0.42ul
dH2O 2.46ul
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 12-15x (1ng)
68C, 5min

Library Quantification and Nextera Preparation[edit]

  • Beads purify once (0.8x) and reconstitute in 10ul
  • Run 1ul on gel:

File:06052014 PCR1 Gel1.jpg



  • Quantify 1ul using Qubit:


Sample Condition [cDNA] ng/ul Volume for 0.2ng/ul Volume dH2O (final 5ul) Nextera Barcode
1 RIII (11) 0.726 1.38 3.62 N503/N701
2 RIII (11) 0.541 1.85 3.15 N503/N702
3 RIII (13) 1.14 0.88 4.12 N503/N703
4 RIII (13) 0.938 1.07 3.93 N503/N704
5 RIII (15) 2.13 0.47 4.53 N503/N705
6 RIII (15) 2.59 0.39 4.61 N503/N706
7 RIII NTC (15) 0.41 2.44 2.56 N503/N707
8 RIII NTC (15) 0.508 1.97 3.03 N503/N708
9 Mg (11) 0.366 - -
10 Mg (11) 0.559 - -
11 Mg (13) 0.52 - -
12 Mg (13) 0.45 - -
13 Mg (15) 1.11 0.90 4.10 N504/N709
14 Mg (15) 0.9 1.11 3.89 N504/N710
15 Mg NTC (15) 0.312 3.21 1.79 N504/N711
16 Mg NTC (15) 0.457 2.19 2.81 N504/N712


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
50x Sybr green 0.08ul
Total 25ul


Mix and centrifuge


72C 3 min
95C 30s
95C 10s -> 55C 30s -> 72C 60s 8x
72C, 5min
  • Run 2.5ul on gel:

File:06042014 PCR2 Gel1.jpg

  • Results: Can see that the reduction of the nextera cycles resulted in a lower cDNA yield compared to 03052014 using 12 cycles - hopefully this will result in fewer clonal arrays in the final libraries
  • Combined 5ul each (10ul for 9 and 10)
  • beads purified twice (0.8x volume)
  • Resuspended in 10ul, ran 1ul on gel:

File:06052014 PCR2 Gel1.jpg