Blue:RNA-Seq Experiments:10102013

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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_LCM_1Cell_N2id_ Mouse Pyramidal 1 cell TSO.r06 T20V.id1 N2.id89 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
BL_LCM_2Cell_N2id_ Mouse Pyramidal 2 cell TSO.r06 T20V.id2 N2.id89 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
BL_LCM_5Cell_N2id_ Mouse Pyramidal 5 cell TSO.r06 T20V.id3 N2.id89 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
BL_LCM_10Cell_N2id_ Mouse Pyramidal 10 cell TSO.r06 T20V.id4 N2.id89 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read
BL_LCM_100pgHC_N2id_ Mouse Pyramidal Handcut(2) - Extraction TSO.r06 T20V.id5 N2.id90 totoRNAseq Read1 plus N2 barcode read and Read2 for T20 barcode read


LCM samples collected 10-09-2013 (10022013 handcut 2 1.4ng/ul)

Purpose[edit]

  • Generate totoRNAseq libraries using RNaseIII digestion on LCM collected samples of mouse brain pyramidal neurons - same as 10032013
  • Determine minimum number of nuclei that can be used for the library preparation

RNAseIII-Fragmented Library Preparation[edit]

Sample Prep
  • Dilute handcut to 133pg/ul (1:10 dilution)
  • Samples collected in 10ul water or Triton-X100
  • Vacuspin to bring volume to ~1.0ul @30C (10-15 minutes?)


Volume
RNA 1.0ul
ERCC 1:10E5 0.2ul
10x RNaseIII Buffer 0.2ul
0.1uM T20V.id 0.1ul
Total 1.5ul
Incubate @ 72C 3 min
Incubate @ 37C 1min
Incubate @ 4C 1min


RNA FRAGMENTATION
Volume
RNA 1.5ul
RNase III 0.08ul
dH20 0.42ul
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.ID2 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

(Note: incubated at 37C by accident - then increased to 42C and re-incubated another 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 10ul dH2O (combine 1-4), 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 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 12x
72C, 5min

Results of Amplification[edit]

  • Run 5ul on gel:


File:10102013 PCR1 gel1.jpg

Final Library Preparation[edit]

  • Beads purify (0.8x), re-suspend in 50ul
  • Beads purify again at 0.8x (if primer dimer band was apparent)
  • Resuspend beads in 10ul
  • Run 1ul on gel: