Kun:LabNotes/SingleCellExpr/2011-7-5: Difference between revisions

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m (Created page with '===Making RNAseq libraries from 100+ cells=== *Use Clonetech's SMARTer Ultra Low RNA kit. *Samples: #Positive control (1ng) #mESC (~50 cells) #mE9.5 (~163 cells) #mE11.5 (~2000 c…')
 
>RuiLiu
 
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*Samples:
*Samples:
#Positive control (1ng)
#Positive control (1ng)
#Negative control (ddH2O)
#YangXu: Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), 20.8ug/ml, use 1ul
#YangXu: Pancreatic lineage differentiated from hESC, NGN3- (~50000 cells, purified with Qiagen RNeasy), 12.7ug/ml, use 1ul
*Will process the following samples later if the first attempt works.
#mESC (~50 cells)
#mESC (~50 cells)
#mE9.5 (~163 cells)
#mE9.5 (~163 cells)
Line 8: Line 13:
#mE13.5 male(~3000 cells)
#mE13.5 male(~3000 cells)
#mE13.5 female(~3000 cells)
#mE13.5 female(~3000 cells)
#YangXu-beta_cell_progenitors (XX cells)
 
====ds-cDNA synthesis====
====ds-cDNA synthesis====
     total RNA                         3.5ul
  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
     3’ SMART CDS Primer II A (12uM)      1ul
    Add ~10ul mineral oil
     72C 3min -> transfer to coldblock
     72C 3min -> transfer to coldblock


Line 27: Line 36:
   Purify the ds-cDNA with AmpPure bead:  
   Purify the ds-cDNA with AmpPure bead:  
     Add 25ul AmpPure beads, mix with pipetting, incubate at RT for 8min;
     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;
     Place the tubes on the magnetic separation device for 5min;
     Pipette out the solution, spin briefly;
     Pipette out the solution, spin briefly;
     Place the tubes back to the magnetic separation device for 2min;
     Place the tubes back to the magnetic separation device for 2min;
     Remove the rest of liquid;
     Remove the rest of liquid;
====PCR amplification====
====PCR amplification====
   Set up QPCR reactions with Kapa HiFi master mix.
   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 (10 cycles for YangXu's samples) -> 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 Qubit), 1ul (left in the tube) for gel check
====PCR products====
TBE PAGE: 1ul; 1ul low mass ladder, 0.5ul ladder, PC, NC, N+, N-
[[File:7.5.11_smarter.jpg]]
[[File:7.5.11_smarter overexposure.jpg]]
Qubit dsDNA HS assay (1ul for concentration, 9ul left in tube):
PC: 0.333ug/ml
NC: too low
N+: 1.59ug/ml
N-: 1.13ug/ml
IS PCR primer: AAGCAGTGGTATCAACGCAGAGT
====Illumina library construction====
====Illumina library construction====
*Covaris shearing to 200bp.
*End-repair
*A-tailing
*TA-ligation
*Enrichment PCR with Kapa HiFi mastermix, and barcoded primers
*Mix and size selection.

Latest revision as of 16:59, 6 July 2011

Making RNAseq libraries from 100+ cells[edit]

  • Use Clonetech's SMARTer Ultra Low RNA kit.
  • Samples:
  1. Positive control (1ng)
  2. Negative control (ddH2O)
  3. YangXu: Pancreatic lineage differentiated from hESC, NGN3+ (~50000 cells, purified with Qiagen RNeasy), 20.8ug/ml, use 1ul
  4. YangXu: Pancreatic lineage differentiated from hESC, NGN3- (~50000 cells, purified with Qiagen RNeasy), 12.7ug/ml, use 1ul
  • Will process the following samples later if the first attempt works.
  1. mESC (~50 cells)
  2. mE9.5 (~163 cells)
  3. mE11.5 (~2000 cells)
  4. mE13.5 male(~3000 cells)
  5. mE13.5 female(~3000 cells)

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                       1ul
    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 (10 cycles for YangXu's samples) -> 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 Qubit), 1ul (left in the tube) for gel check

PCR products[edit]

TBE PAGE: 1ul; 1ul low mass ladder, 0.5ul ladder, PC, NC, N+, N-

File:7.5.11 smarter.jpg

File:7.5.11 smarter overexposure.jpg

Qubit dsDNA HS assay (1ul for concentration, 9ul left in tube):
PC: 0.333ug/ml
NC: too low
N+: 1.59ug/ml
N-: 1.13ug/ml
IS PCR primer: AAGCAGTGGTATCAACGCAGAGT

Illumina library construction[edit]

  • Covaris shearing to 200bp.
  • End-repair
  • A-tailing
  • TA-ligation
  • Enrichment PCR with Kapa HiFi mastermix, and barcoded primers
  • Mix and size selection.