Blue:RNA-Seq Experiments:09182013

From ZhangLabWiki
Jump to navigation Jump to search

RNA-Seq Library Information[edit]

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Type of Seq Reads Library Conc
BL_UHRR_ UHRR/ERCC 1ng - Unfragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter Read 1 and N2 barcode read
BL_UHRR_ UHRR/ERCC 100pg - Unfragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter
BL_UHRR_ UHRR/ERCC 10pg - Unfragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter
BL_UHRR_ UHRR/ERCC 1ng - Unfragmented-PolyA STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter Full-length totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg - Unfragmented-PolyA STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter Full-length totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - Unfragmented-PolyA STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter Full-length totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg - RNaseIII Fragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - RNaseIII Fragmented STRT_V2_3 (In-house) STRT_V3_T30 (In-house) Smarter totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented STRT_V2_3 (In-house) T20V.id N2_id9 totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg - RNaseIII Fragmented STRT_V2_3 (In-house) T20V.id N2_id10 totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - RNaseIII Fragmented STRT_V2_3 (In-house) T20V.id N2_id11 totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RNaseIII Fragmented - 1 step TSO_r03 T20V.id N2_id12 totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg - RNaseIII Fragmented - 1 step TSO_r03 T20V.id N2_id13 totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg - RNaseIII Fragmented - 1 step TSO_r03 T20V.id N2_id14 totoRNAseq

Purpose[edit]

  • Use variations of our protocol to evaluate the quality of library generation using Smart primers (kit or in-house) using ERCC QC assay
  • Examine efficiency of Smarter protocol (unfragmented) compared to Poly-A tailing (unfragmented) and fragmentation using ERCC QC assay
  • Examine efficiency of the modified TSO r03 (resistant to Poly-A tailing) in a single step totoRNAseq reaction using ERCC QC assay

Library Preparation (Unfragmented)[edit]

RNA PREPARATION
Volume
2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
STRT_V3_T30(10uM) 1.0ul
dNTP (10mM) 1ul
dH2O 1.8ul
Total 4.5ul


Incubate @ 65C 3 min
Transfer to ice


Volume
5xRT Buffer 2ul
100mM DTT 0.17ul
STRT_V2_3 TSO (10uM) 1ul
Betaine 2ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (100U/ul) 0.23ul
Total 10ul


Incubate @ 42C 90 min
Incubate @ 70C 10 min
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 10ul dH2O, incubate 2min, transfer to new tube


PCR Amplification
Volume
cDNA 10ul
2x KAPA HiFi Hotstart ReadyMix 25ul
(10uM) STRT_PCR 1.0ul
dH2O 14ul
Total 50ul


  • 98C 3min
  • 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
  • 98C 15 sec
  • 67C 20sec
  • 72C 6min
  • 72C 5min

Poly-A Unfragmented Library Preparation[edit]

RNA PREPARATION
Volume
2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
5x RT Buffer 0.5ul
1mM ATP 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul
dH20 0.8ul
Total 2.5ul


Incubate @ 37C 5 min
Transfer to Ice


REVERSE TRANSCRIPTION
Volume
PAP RNA 2.5ul
10mM dNTP Mix 1ul
10uM STRT_V3_T30 1ul
dH2O 1.5ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 1.5ul
100mM DTT 0.17ul
Betaine 2ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (200U/ul) 0.23ul
Total 10ul


Incubate @ 42C 90 min


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 to 3ul


TSO
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


USER
Add 1ul and incubate at 37C for 30 min



PCR Amplification
Volume
cDNA 10ul
2x KAPA HiFi Hotstart ReadyMix 25ul
STRT_PCR (10mM) 1.0ul
dH2O 14ul
Total 50ul


  • 98C 3min
  • 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
  • 98C 15 sec
  • 67C 20sec
  • 72C 6min
  • 72C 5min

RNAseIII-Fragmented Library Preparation[edit]

RNA PREPARATION
Volume
2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
10x RNaseIII Buffer 0.2ul
1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30 0.1ul
dH20 0.25ul
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 1.25ul
RNase III 0.08ul
dH20 0.67ul
Total 2ul


Incubate @ 37C 10 min
Add 1.1ul dH2O
Incubate @ 65C 10 min


3' Tailing
Volume
fRNA 3.1ul
5x RT Buffer 0.5ul
1mM ATP 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul


Incubate @ 37C 5 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 4.1ul
10mM dNTP Mix 1ul
1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 or STRT_V3_T30 0.9ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 1.5ul
100mM DTT 0.17ul
Betaine (5M) 2ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (200U/ul) 0.23ul
Total 10ul


Incubate @ 42C 30 min


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 to 3ul


TSO
Volume
Purified cDNA 3ul
5x RT buffer [MMLV] 2ul
10mM dNTP mix 1ul
100mM DTT 0.25ul
10uM STRT_V2_3 TSO 1ul
Betaine (5M) 2ul
RNase Inhibitor 0.25ul
Smarter MMLV 0.5ul
Total 10ul


Incubate @42C 10min
Incubate @70C 10min


USER
Add 1ul and incubate at 37C for 30 min


PhusionHF PCR (for T20V.ID)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM P1-STRT 1ul
10uM PCR_R_N2_id 1ul
H2O 12ul
Total 50ul


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


KAPAHF PCR (STRT_V3_T30)
Volume
cDNA 11ul
2x KAPA HiFi Hotstart ReadyMix 25ul
STRT_PCR (10mM) 1.0ul
dH2O 13ul
Total 50ul


  • 98C 3min
  • 12 cycles (1ng), 15 cycles (100pg) or 18 Cycles (10pg):
  • 98C 15 sec
  • 67C 20sec
  • 72C 30sec
  • 72C 5min

RNAseIII-Fragmented Library Preparation (1-Step)[edit]

RNA PREPARATION
Volume
2ng/ul, 200pg/ul, 20pg/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
10x RNaseIII Buffer 0.15ul
1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 0.1ul
Total 0.95ul
Incubate @ 72C 3 min
Incubate @ 37C 1min


RNA FRAGMENTATION
Volume
RNA 0.95ul
RNase III 0.08ul
dH20 0.47ul
Total 1.5ul


Incubate @ 37C 10 min
Add 0.6ul dH2O
Incubate @ 65C 10 min


3' Tailing
Volume
fRNA 2.1ul
5x RT Buffer 0.5ul
1mM ATP 0.25ul
0.5U/ul PAP (1:10d in H2O) 0.25ul


Incubate @ 37C 5 min
Transfer to Ice


Reverse Transcription
Volume
PAP RNA 3.1ul
10mM dNTP Mix 1ul
1uM(1ng) or 0.1uM(100pg/10pg) T20V.id41 0.9ul


Incubate @ 65C 3 min
Transfer to Ice


Volume
5xRT Buffer 1.5ul
100mM DTT 0.17ul
Betaine (5M) 2ul
TSO.r03 (10uM) 1ul
RNase Inhibitor (40U/ul) 0.1ul
Smarter MMLV (200U/ul) 0.23ul
Total 10ul


Incubate @42C 30 min
Incubate @70C 10 min


USER
Add 1ul and incubate at 37C for 30 min


PCR (PhusionHF)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM PB_PCR_F 1ul
10uM PCR_R_N2_id 1ul
H2O 12ul
Total 50ul


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

Results of Amplification[edit]

  • Run 5ul on gel


File:09182013 PCR1 gel1.jpg

ERCC Quality Control Analysis[edit]

  • Comparison of Pre-amp and post-amp plots:
    • ERCC-130-5'F/R
    • ERCC-130-midF/R
    • ERCC-130-3'F/R
  • Controls (carried through RT and Beads purification but no TSO/PCR - made up to 50ul):
    • Unfragmented STRT
    • Unfragmented Smart
    • PolyA Unfragmented STRT
    • Fragmented STRT
    • Fragmented T20V


  • Amplified 1ul control and 1ul PCR (total volume 25ul) using Sybr-Green:
Volume
Library 1ul
2xSybr Buffer 12.5ul
10uM F primer 0.5ul
10uM R Primer 0.5ul
dH2O 10.5ul
Total 25ul


95C 30sec
95C 10s -> 60C 20s -> 72C 30s => 40X
72C, 5min

Loading Format for qPCR:

' ' Smart ' ' Smart-PolyA ' ' Smart-Frag ' ' Smart-T20 '
5' mid 3' 5' mid 3' 5' mid 3' 5' mid 3'
1ng PCR Smart Smart Smart Smart-PolyA Smart-PolyA Smart-PolyA Smart-Frag Smart-Frag Smart-Frag Smart-T20 Smart-T20 Smart-T20
100pg PCR Smart Smart Smart Smart-PolyA Smart-PolyA Smart-PolyA Smart-Frag Smart-Frag Smart-Frag Smart-Frag Smart-T20 Smart-T20
10pg PCR Smart Smart Smart Smart-PolyA Smart-PolyA Smart-PolyA Smart-Frag Smart-Frag Smart-Frag Smart-Frag Smart-T20 Smart-T20
1ng RT Smart Smart Smart Smart-PolyA Smart-PolyA Smart-PolyA Smart-Frag Smart-Frag Smart-Frag Smart-Frag Smart-T20 Smart-T20
100pg RT Smart Smart Smart Smart-PolyA Smart-PolyA Smart-PolyA Smart-Frag Smart-Frag Smart-Frag Smart-Frag Smart-T20 Smart-T20
10pg RT Smart Smart Smart Smart-PolyA Smart-PolyA Smart-PolyA Smart-Frag Smart-Frag Smart-Frag Smart-Frag Smart-T20 Smart-T20


File:09182013 ERCC130 Smart.jpg

Note: second chart compares 3' amplification for ERCC130 at different [RNA]



File:09182013 ERCC130 Smart2.jpg

File:09182013 ERCC130 Smart3.jpg

Final Library Preparation[edit]

  • Beads purified (0.8x)
  • Re-amplified whole libraries 6x (1ng) or 7x (100pg, 10pg)
  • Beads purified (0.8x)
  • Ran 1ul on gel:

File:09182013 PCR2 gel1.jpg

Notes:

  • Library amplifications seemed low - might be an issue with one of the reagents e.g. MMLV. Therefore, need to repeat library production
  • Combined Smart/r03 (id9/12) (1ng seen above) for sequencing