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==Making RNAseq library from 100+ cells== * Continue with lab note [http://genome-tech.ucsd.edu/LabNotes/index.php/Kun:LabNotes/SingleCellExpr/2011-7-5#PCR_products] ===Samples=== * 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: # Positive control (1ng) # 1 tube of mE9.5 (~316 cells; ~3ng) # 1/10V of mE11.5 [2ul out of 20ul] (~2000 cells; ~20ng) # 1/10V of mE13.5 male [2.5ul out of 25ul](~3000 cells; ~30ng) # 1/10V of mE13.5 female [5ul out of 50ul] (~3000 cells; ~30ng) ===Procedure=== ====RNA isolation==== * 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==== # 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==== Make reaction buffer: 19ul dilution buffer + 1ul RNase Inhibitor => 20ul RNA denaturing and annealing: Reaction buffer 2.5ul total RNA 1ul 3β 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==== 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==== 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]]
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