Blue:RNA-Seq Experiments:10012013

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RNA-Seq Library Information

Library ID Samples Input RNA TSO Primer Bar Code Primers PCR Primers Number of Cycles Amp Type of Seq Reads Library Conc
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented 10 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id1 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented 5 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id2 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg (x2) - RNaseIII Fragmented 10 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id7-8 18 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg (x2) - RNaseIII Fragmented 5 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id9-10 18 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg (x2) - RNaseIII Fragmented 10 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id11-12 21 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg (x2) - RNaseIII Fragmented 5 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id13-14 21 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 10 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id3 15 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 5 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id4 15 Cycles totoRNAseq
BL_HuLCM_ HuLCM/ERCC 1ng - RnaseIII Fragmented 10 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id5 15 Cycles totoRNAseq
BL_HuLCM_ HuLCM/ERCC 1ng - RnaseIII Fragmented 5 min TSO_r04_N6 T20V.id1 PB_PCR_F/N2_id6 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented 10 min TSO.r06 T20V.id1 P1-STRT/N2_id15 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 1ng - RnaseIII Fragmented 5 min TSO.r06 T20V.id1 P1-STRT/N2_id16 15 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg (x2) - RNaseIII Fragmented 10 min TSO.r06 T20V.id1 P1-STRT/N2_id21-22 18 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 100pg (x2) - RNaseIII Fragmented 5 min TSO.r06 T20V.id1 P1-STRT/N2_id23-24 18 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg (x2) - RNaseIII Fragmented 10 min TSO.r06 T20V.id1 P1-STRT/N2_id25-26 21 Cycles totoRNAseq
BL_UHRR_ UHRR/ERCC 10pg (x2) - RNaseIII Fragmented 5 min TSO.r06 T20V.id1 P1-STRT/N2_id27-28 21 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 10 min TSO.r06 T20V.id1 P1-STRT/N2_id17 15 Cycles totoRNAseq
BL_HuNu_ HuNu/ERCC 1ng - RnaseIII Fragmented 5 min TSO.r06 T20V.id1 P1-STRT/N2_id18 15 Cycles totoRNAseq
BL_HuLCM_ HuLCM/ERCC 1ng - RnaseIII Fragmented 10 min TSO.r06 T20V.id1 P1-STRT/N2_id19 15 Cycles totoRNAseq
BL_HuLCM_ HuLCM/ERCC 1ng - RnaseIII Fragmented 5 min TSO.r06 T20V.id1 P1-STRT/N2_id20 15 Cycles totoRNAseq


Primer List

UHRR=Universal Human Reference RNA; ERCC=spike in RNA; HuNu = RNA from Human Pyramidal Nuclei sorted 09-23-13; HuLCM = RNA from bulk Human Pyramidal LCM sample (File:ERCC bioanalyzer 9.18.13.pdf)

Purpose

  • Generate totoRNAseq libraries using modified Smarter TSO (r06) and modified r04 TSO to compare efficiencies
  • Compare TSO efficiencies over different RNA amounts and between UHRR and human pyramidal RNA generated from sorted nuclei or LCM
  • Compare RNAseIII fragmentation times: full (10min) or partial (5min)

RNAseIII-Fragmented Library Preparation

RNA PREPARATION
Volume
2ng/ul, 200ng/ul, 20ng/ul UHRR 0.5ul
ERCC 1:10E4, 1:10E5, 1:10E6 0.2ul
10x RNaseIII Buffer 0.2ul
1uM or 0.1uM (10-100pg) T20V.id1 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 5-10 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 or 0.1uM (10-100pg) 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
  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 5 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


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


PCR (PhusionHF)
Volume
TSO Reaction 11ul
2x Buffer 25ul
10uM P1-STRT (Smarter TSO) or 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 9x (1ng), 12x (100pg), 15x (10pg)
72C, 5min

Results of Amplification

  • Run 5ul on gel:

File:09272013 PCR1 gel1.jpgFile:09272013 PCR1 gel2.jpg

Note: U=UHRR; H=Sorted Human Nuclei; L=LCM Human Nuclei; 10=10 minutes RNaseIII fragmentation; 5=5 minutes RNaseIII fragmentation


File:09272013 PCR1 gel3.jpgFile:09272013 PCR1 gel4.jpg

Final Library Preparation