Rui:LabNotes/Collaborations/2011-7-6

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Making RNAseq library from 100+ cells[edit]

  • Continue with lab note [1]

Samples[edit]

  • Use ZR RNA MicroPrep (Zymo R1060) for RNA isolation from cells (The protocol of this kit is different from the first kit I used before.)
  • Use Clonetech's SMARTer Ultra Low RNA kit for RNA library prep.
  • Samples:
  1. Positive control (1ng)
  2. 1 tube of mE9.5 (~316 cells; ~3ng)
  3. 1/10V of mE11.5 [2ul out of 20ul] (~2000 cells; ~20ng)
  4. 1/10V of mE13.5 male [2.5ul out of 25ul](~3000 cells; ~30ng)
  5. 1/10V of mE13.5 female [5ul out of 50ul] (~3000 cells; ~30ng)

Procedure[edit]

RNA isolation[edit]

  • Change highlighted in bold
  1. Add 400ul RNA lysis buffer to samples and mix well
  2. Centrifuge the sample mixture at >12kg for 1min
  3. Transfer the lysate to a Zymo-Spin IIIC column in a collection tube, spin at 8kg for 30sec
  4. Add 0.8V ethanol to flow-through in the collection tube and mix well (320ul to 400ul)
  5. Transfer the mixture to a Zymo-Spin IC Column in a collection tube, spin at 12kg for 1min, discard the flow-through
  6. Pre-make the following DNaseI cocktail for each sample: 10X buffer 3ul; DNase I 3ul; RNA wash buffer 24ul
  7. Add 400ul RNA wash buffer to Zymo-Spin IC Column in a collection tube, spin at 12kg for 30sec, discard the flow-through
  8. Add 30ul DNase I (stored at -20C, box1) cocktail directly to the matrix of the Zymo-Spin IC Column , incubate at 25-37C > 15min
  9. Spin >12kg for 30sec, discard the flow-through
  10. Add 400ul RNA prep buffer to the column, spin >12kg for 1min, discard the flow-through
  11. Add 800ul RNA wash buffer to the column, spin >12kg for 30sec, discard the flow-through, repeat with 400ul RNA wash buffer
  12. Spin > 12kg for add. 2min
  13. Place in a tube, add 20ul water to matrix, RT for 1min
  14. Spin 10kg for 30sec

RNA precipitation[edit]

  1. RNA sample above, 2ul 3M NaOAc, 50ul 100% EtOH, 2ul Glycoblue (15 mg/ml) [optional, b/c the pellet is visible.]
  2. -80C for 30min; spin 4C 11k rpm for 30min; wash; air-dry; re-suspend in 1ul

ds-cDNA synthesis[edit]

  Make reaction buffer: 19ul dilution buffer + 1ul RNase Inhibitor => 20ul
  RNA denaturing and annealing:
    Reaction buffer                    2.5ul
    total RNA                            1ul
    3’ SMART CDS Primer II A (12uM)      1ul
    Add ~10ul mineral oil
    72C 3min -> transfer to coldblock
  Prepare master mix:
    5X First-Strand Buffer                       2ul
    100mM DTT                                 0.25ul
    dNTP Mix                                     1ul
    SMARTer II A Oligonucleotides (12uM)         1ul
    RNase Inhibitor                           0.25ul
    SMARTScribe Reverse Transcriptase (100U)     1ul
  Add 5.5ul master mix to each reaction, mix by gentle pipetting, spin briefly.
    42C 90min -> 70C 10min
  Purify the ds-cDNA with AmpPure bead: 
    Add 25ul AmpPure beads, mix with pipetting, incubate at RT for 8min;
    Briefly spin-down to separate mineral oil/aqueous; 
    Place the tubes on the magnetic separation device for 5min;
    Pipette out the solution, spin briefly;
    Place the tubes back to the magnetic separation device for 2min;
    Remove the rest of liquid;

PCR amplification[edit]

  Set up QPCR reactions with Kapa HiFi master mix.
    2X Master Mix        25ul
    IS PCR primer (12uM)  2ul
    H2O                  23ul
  95C 2min -> (98C 20sec -> 65C 15sec -> 72C 2min) x 12 cycles (17 cycles) -> 72C 3min.
  Purify the dsDNA with AmpPure bead: 
    Add 90ul AmpPure beads, mix with pipetting, incubate at RT for 8min;
    Place the tubes on the magnetic separation device for 5min;
    Pipette out the solution, wash with 200ul 80% EtOH, repeat wash one more time
    Seal tubes and briefly spin down, remove the rest of liquid;
    Air-dry 3-5min;
    Resuspend in 12ul purification buffer; take out 10ul (1ul for gel), 1ul (left in the tube for 3 samples) for Qubit


PCR results[edit]

Qubit: E9.5 2ng/ul; E11.5 7ng/ul; E13.5 3ng/ul

1ul for TBE PAGE

File:7.7.11 SMARTer.jpg

overexposure for possible contamination


File:7.7.11 SMARTer over.jpg