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 |
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
- Add 15ul beads per well
- Allow to bind 15 min
- Remove supernatant and wash 2x with 1ml 80% EtOH
- Air Dry 10min or until cracking first occurs
- 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