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
- Add 400ul RNA lysis buffer to samples and mix well
- Centrifuge the sample mixture at >12kg for 1min
- Transfer the lysate to a Zymo-Spin IIIC column in a collection tube, spin at 8kg for 30sec
- Add 0.8V ethanol to flow-through in the collection tube and mix well (320ul to 400ul)
- Transfer the mixture to a Zymo-Spin IC Column in a collection tube, spin at 12kg for 1min, discard the flow-through
- Pre-make the following DNaseI cocktail for each sample: 10X buffer 3ul; DNase I 3ul; RNA wash buffer 24ul
- Add 400ul RNA wash buffer to Zymo-Spin IC Column in a collection tube, spin at 12kg for 30sec, discard the flow-through
- Add 30ul DNase I (stored at -20C, box1) cocktail directly to the matrix of the Zymo-Spin IC Column , incubate at 25-37C > 15min
- Spin >12kg for 30sec, discard the flow-through
- Add 400ul RNA prep buffer to the column, spin >12kg for 1min, discard the flow-through
- Add 800ul RNA wash buffer to the column, spin >12kg for 30sec, discard the flow-through, repeat with 400ul RNA wash buffer
- Spin > 12kg for add. 2min
- Place in a tube, add 20ul water to matrix, RT for 1min
- Spin 10kg for 30sec
RNA precipitation[edit]
- RNA sample above, 2ul 3M NaOAc, 50ul 100% EtOH, 2ul Glycoblue (15 mg/ml) [optional, b/c the pellet is visible.]
- -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