Blue:RNA-Seq Experiments:PAP opt

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RNA-Seq Library Information

Library ID Samples Input RNA TSO Primer Bar Code Primers Bar Code Primer 2 Type of Seq Reads Library Conc
BL_ UHRR/ERCC 1ng - RNase3 TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 1ng - RNase3 TSO.r06 T30.SE.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 100pg - RNase3 TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 100pg - RNase3 TSO.r06 T30.SE.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 TSO.r06 T30.SE.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 2 min TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 4 min TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 6 min TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 - 0.01/1 Cordycepin TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 - 0.05/1 Cordycepin TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 - 0.1/1 Cordycepin TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read
BL_ UHRR/ERCC 10pg - RNase3 - 0.05/1 Cordycepin TSO.r06 T20V.id N2.id] totoRNAseq Read1 plus N2 barcode read


Purpose

  • Test T20 versus T30
  • Test RNaseIII fragmentation times
  • Test PAP inactivation - heating
  • Test PAP termination Cordycepin-ATP


RNAseIII-Fragmented Library Preparation

Cell Sort
Volume
RNA 250pg or Single Nuclei -
1x Lysis Buffer 0.75ul
Total 0.75ul

Note: add in UHRR control at this point


Lysis
Volume
RNA 0.75ul
10x RNaseIII Buffer 0.2ul
0.1uM T20V.id 0.1ul
dH20 0.2ul (1250pg/ul UHRR for 250pg or 50pg/ul for 10pg)
Total 1.25ul
Incubate @ 72C 3 min
Incubate @ 37C 1min
Incubate @ 4C 10min


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
0.1uM T20V.ID 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, combine
  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 - vacuspin 8 min to bring 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


PCR (PhusionHF)
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 12x
72C, 5min

Results of Amplification

  • Run 5ul on gel:

File:10162013 PCR1 gel1.jpg

Final Library Preparation

  • Beads purified twice at (0.8x), re-suspend in 10ul
  • Re-Amplified using ILMN_PCR_F/R:
  1. huNu (~30) id81 = 2ul original PCR
  2. UHRR (250pg) = 0.5ul original PCR
  3. NC
  • Ran 5ul (of 50ul) on gel:

File:10162013 PCR2 gel1.jpg


  • Clearly the huNu library did not work...