If using cells which grow in suspension, spin at 500xG for 5 minutes.
Remove media by aspiration or by dumping into a waster container.
Add 100uL of cold 1xPBS with a multichannel to every well to wash off residual media.
Remove cold 1xPBS from wells via aspirating or dumping into a waste container.
Add 50uL of Tryp-LE per well and place plate into a 37C incubator.
Allow to incubate for 10-20 minutes based on the cell line.
After cells are properly detached, add 150uL of cell culture medium to quench the reaction.
Using a new set of tips for every well, transfer the 200uL volume cell suspension into a V-bottom 96 well plate. Make sure that the orientation is preserved between the 96 well culture plate and the 96 well V-bottom plate.
Spin for 5 minutes at 300xG to pellet cells.
Remove media by aspiration or by dumping into a waster container.
Add 100uL of cold 1xPBS with a multichannel to each well to wash off residual media.
Spin cells down again at 300xG for 5 minutes at 4C in the V-bottom plate to pellet cells.
NOTE: Keep sample on ice and spin at 4C whenever possible from here on.
Add 50uL of ice-cold CLB solution to each well of the 96 well V-bottom plate using a wide bore tip. Make sure to resuspend and mix using 3-5 strokes. Discard tips when finished.
Hashing: To each well add 1 uL 10uM Hash transferred with preserved orientation from 96 well plate. The amount of hash added should be adjusted based on the number of cells/nuclei in each well. We typically target 0.5 pmol hash per 1000 cells. Adding too little hash can reduce the labeling efficiency.
Hash oligos have the sequence: 5’-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXBAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’, where ‘X’s represent the 10nt, well-specific hash ID.
Stir with pipette tip to mix (do not pipette up and down).
Incubate on ice for 5 minutes.
Using a multichannel with wide-bore tips add 108uL of fixation buffer. Mix with 3 strokes to ensure complete permeation of the fixative.
Incubate plate for 15 minutes on ice.
Spin down cells for 5 minutes at 500xG at 4C.
Pool all wells of fixed nuclei into 15ml conical.
Pellet nuclei for 5 minutes at 500xG at 4C.
Resuspend in 1mL of Nuclei Buffer with 0.1% Tween 20.
Allow to rest on ice for 3 minutes.
Spin down at 500xG for 5 minutes.
Resuspend in 1mL of nuclei buffer.
Spin down at 500xG for 5 minutes at 4C.
Resuspend in 0.5 mL Nuclei Buffer for counting.
take 5-10uL of resusupended nuclei, mix with equal volume of trypan blue, count cells and bring to final concentration of 2.5x10e6 cells/mL in Nuclei Buffer.