Rui:LabNotes/Collaborations/2011-10-3

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Making RNAseq library[edit]

Samples[edit]

  • 6 samples from Dr. Yi Zhang lab [1] on 9.15.11
  • 3 samples from Dr. Xu Yang lab [2] on 7.7.11
  • 3 samples from Dr. Xu Yang lab by Alice on 9.28.11: RNA samples from sorted neural stem cells from nestin-rfp knockin hESCs. Low is rfp low, high rfp high, fetal NSCs are the human fetal cells.

Procedure[edit]

  • To save the reagent, I used some old ones from August (probably a bad idea!)
  • Due to 12 samples instead of 10, the PCR primer is roughly 1 or 2 ul less than it should be (24ul)
  • I did 15 cycles of PCR instead of 17 cycles this time.
  • 3 neuronal RNA samples are 7ng (#10), 5ng (#11), and 3ng (#12)

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 15 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]

cDNA libraries[edit]

Dr. Yi Zhang lab samples:

  • When carefully check the lab note before, I feel like 4 samples (s1, 3, 4, 5) are actually consistent with previous data - 2 cycles less (roughly 4 fold difference) and less cell numbers (1000c).
  • However, s2 and s6 are ~5 fold lower than it should be. As I processed all samples together, it probably suggests the cell number was not accurate in these 2 tubes or the quality of cells was not good.

Dr. Xu Yang lab samples:

  • I am a bit concerned about this set of cells Dae-Hwan gave me, because he did several rounds of spinning at top speed for 10min each (I mentioned this on main page of lab note), especially for s8 (RFP+) which had the least cell number.
  • For the last 3 RNA samples, Alice told me those are quite old samples sitting in freezer for a while. I need to double check the concentration to see if it is consistent with the label. I took 7ng (1ul) for s10, 5ng (1ul) for s11 and 3ng (1ul of 1:100d) for s12.
Sample concentration ' total amount
1 1.07 ng/ul 10.7
2 0.2 ng/ul 2.01
3 1.31 ng/ul 13.1
5 0.67 ng/ul 6.73
6 0.12 ng/ul 1.2
8 0.31 ng/ul 3.09
9 1.57 ng/ul 15.7
10 1.49 ng/ul 14.9
11 2.68 ng/ul 26.8
12 1.33 ng/ul 13.3

Libraries 1-6 (Dr. Yi Zhang lab) File:10.4.11 1-6.jpg Libraries 7-12 (Dr. Xu Yang lab) File:10.4.11 7-12.jpg