2.3 Post Transposition Wash

  1. Prepare Transposition Wash Buffer on ice. Pipette mix 10x and centrifuge briefly.

     

    Transposition Wash Buffer

    Add reagents in the order listed

     

    PN

     

    1X (µl)

    4X +

    10% (µl)

    8X +

    10% (µl)

    20X Nuclei Buffer

    2000207

    		 9.0 39.6 79.2

    Reducing Agent B

    200087

    		 0.4 1.8 3.5

    RNase Inhibitor
    (use ONLY PN-2001488)

    2001488

    		 7.6 33.4 66.9
     

    Nuclease-Free Water

    		 - 283.0 1245.2 2490.4
     

    Total

    -

    300

    1,320

    2,640

  2. Add 200 μl Transposition Wash Buffer to each sample. Maintain on ice.

  3. Gently pipette mix 10x.

    Remaining Transposition Wash Buffer can be maintained on ice for dilutions in GEM Generation step.

  4. Place the tube strips on plates and use plate adapters to centrifuge at 500 rcf for 5 min at 4°C.

    Using a swinging bucket centrifuge can increase nuclei recovery during centrifugation. During centrifugation, Master Mix may be prepared as outlined in Prepare Master Mix.

  5. Using a pipette tip slightly submerged in the supernatant, start aspirating 190 μl supernatant. When aspirating, continue to keep the pipette tip slightly submerged by moving it down the tube along with the supernatant, leaving behind ~35 μl supernatant in the original tube along with the pellet. The pellet may not be always visible.

    If uncertain about the volume remaining in the tube, add 35 μl water/PBS to an empty tube and use it for a visual estimation.

  6. Resuspend the pellet in the residual 35 μl supernatant (pipette mix 20x) and maintain the tube on ice.

    Use 5 μl nuclei suspension to count and determine nuclei concentration and then proceed to GEM Generation.

    5-10 μl may be used for counting depending on specific sample and counting method. For accurate nuclei counting guidelines, consult sample preparation protocols CG000365 Rev E or CG000505 Rev B.