Blue:RNA-Seq Experiments:01222014

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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_totoRNAseq_10pgUHRR_Jan22 UHRR/ERCC 10pg - 0.1 RNase3 1min/Affymetrix PAP 4min TSO.r06 T20VN_id1-10 N2.id81 totoRNAseq
UHRR/ERCC 10pg - 0.05 RNase3 1min/Affymetrix PAP 4min TSO.r06 T20VN_id1-10 N2.id82 totoRNAseq
UHRR/ERCC 10pg - 0.01 RNase3 1min/Affymetrix PAP 4min TSO.r06 T20VN_id1-10 N2.id83 totoRNAseq
UHRR/ERCC 1ng - 0.1 RNase3 1min/Affymetrix PAP 10min TSO.r06 T20VN_id1 N2.id84-87 totoRNAseq
UHRR/ERCC 100pg - 0.1 RNase3 1min/Affymetrix PAP 8min TSO.r06 T20VN_id1 N2.id88-91 totoRNAseq
  • Note: 10pg/0.01U T20VN.id1 was actually 0.1U RNase3

Primer sequences [1]

Purpose[edit]

  • Given the difficulty in concluding optimal conditions from individual 10pg libraries, will test different RNaseIII treatment conditions on combined 10pg UHRR libraries
  • Previous RNaseIII optimizations involved reducing the time for this reaction down to 15s, but this is quite short and might still not be optimal. Will keep the reaction time constant and try reducing the [RNaseIII]
  • Use ERCCs to determine level of transcript amplification between libraries
  • Combine libraries prior to TSO and amplification to reduce variability between samples and amplification cycle number
  • Compare with single cell sorted samples ([2])


RNAseIII-Fragmented Library Preparation[edit]

RNA
Volume
2ng/ul or 0.02ng/ul UHRR 0.5ul
1:10E4 or 1:10E6 ERCC 0.2ul
0.1uM T20VN 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, 0.05ul (+ 0.05ul dH2O), or 0.1ul of 1:10 dil
dH20 0.7ul
Total 2ul


Incubate @ 37C 1min


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, 8 or 10 min
Add 1ul of 0.1uM T20VN


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 @ *37C 30 min + 30 min
  • accidentally incubated at 37C then extended reaction to be safe


Beads Purification
  1. Add 15ul beads per well
  2. Allow to bind 15 min, combine wells
  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 Reaction
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 10ul
2x Buffer 25ul
10uM P1-STRT 1ul
10uM PCR_R_N2_id 1ul
H2O 13ul
Total 50ul


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

Results of Amplification[edit]

  • Run 5ul on gel:

File:01222014 PCR1 Gel1.jpg

Results: Similar smear amplification for all conditions

Final Library Preparation[edit]

  • Beads purified twice at (0.8x), re-suspended in 10ul
  • PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964.

File:01222014 ERCC130 R227.jpgFile:01222014 ERCC130 R964.jpg


  • Results: ERCC amplication for the combined 10pg libraries was more similar to the 1ng in this experiment. 100pg libraries showed poorer amplification (not shown) in comparison - might be due to the reduction of PAP from 10 min to 8 min - this may not be optimal. Comparing the different amounts of RNase3 (0.1-0.01U), we can see that the 0.1U actually showed similar amplifications to the 1ng replicates, while 0.01U units was less effective. Interestingly, the 0.05U actually seemed to be improved over the 1ng condition. Therefore, 1 minute incubation with 0.05U RNase3 might be the best condition for 10pg. Also, its possible that the 1 hour RT reaction at 37C improved the outcome for these lower inputs. Therefore, will need to generate similar libraries at 42C for comparison.


Final library gel image