Alice:LabNotes/2011-5-11

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DNA accessibility assay on small number of cells[edit]

Idea[edit]

  • Performed Nextera tagmentation on cell lysates with and without proteinase digestion. The idea is that for cells that lysed with mild buffer, histones are still attached to the DNA and most likely prevent tagmentation. Therefore, the presence of tagmentation sites on the genome would indicate regions free of histones.

Experiment protocol[edit]

  • Make 10ml 10X lysis buffer (LB, 100mM Tris.Hcl pH 7.5, 100mM NaCl, 30mM MgCl2, 1% NP40, Crawford et al. PNAS 2004).
  • Prepare aliquots of lymphocytes (GM20431) that contain 500 cells, 200 cells, and 100 cells in 1ul.
  • Prepare 2X LB from 10X buffer with and without Qiagen protease (0.1AU/ml). Add one drop (~10ul) of mineral oil.
  • Set up another group of controls using another 2x lysis buffer (LB2) that Dr. Zhang prepared on 8/18/2010 (20M EDTA, 10mM Tris.HCl , 200mM NaCl, 0.2% Triton X-100, 0.1AU/ml Qiagen Protease), which could remove DNA binding proteins more completely.
  • Mix 1ul cell solution with 1ul lysis buffer
                     A          B          C          D          E           F        K        G          H          I        J 
                 500 cells  200 cells  100 cells   500 cells  200 cells  100 cells   NTC   500 cells  200 cells  100 cells   NTC   
 cells              1ul        1ul        1ul         1ul       1ul         1ul    1ul(PBS)   1ul        1ul        1ul    1ul(PBS)
 2x LB              1ul        1ul        1ul         -          -          -         -        -          -          -        - 
 2x LB (Protease)    -          -         -           1ul       1ul         1ul       1ul      -          -          -        - 
 LB2                 -          -         -           -         -            -        -       1ul        1ul        1ul      1ul
  • Incubate at 37C for 30min. For D-K, proceed with heat inactivation at 75C for 15min.
  • Perform tagmentation.
 Dilute the enzyme: 1:10     
 For each rxn, used mix of: 
  1ul 5x LMW Buffer
  2ul cell lysate
  1ul diluted enzyme
  2ul H2O
-----------------------
  6ul total / reaction
 55C 10 min
  • Set up PCR
  Tagmentation reaction    10ul
  KAPA QPCR mix            25ul
  Orange Primer (10uM)      1ul
  Blue Primer (10uM)        1ul
  Bst Pol (5U/ul)         0.5ul
  H2O                      13ul
  65C 3min -> 95C 30 sec -> (95C 10sec -> 58C 30 sec -> 72 1min) x 22 -> 72C 3min.
  Monitor the reactions on a real-time thermal cycler and terminate them before the curves reach saturation.
  • Sample D was stopped at cycle 20 since it already reached plateau, and the rest of the samples were stopped at cycle 22
  • Ampure bead purification and elute in 40ul ddH2O
  • PAGE result:

File:ZhangLab 2 2011-05-12 13hr 01min.jpg

Discussion[edit]

  • The PAGE gel results suggest that the new buffer is working well, and the results came out as expected, which the cells lysed with protease were more effective than lysed with buffer alone. The old buffer didn't seem to work well, which wasn't what we expected. However, it is possible that since the buffer was prepared a while back, so it doesn't work as effective as fresh made ones. We included the old buffers for comparison purposes, so we will just not include them in the sequencing libraries.