Spin down culture plate (300xG at for 5 minutes room temperature) (e.g. Cells grown in 96-well format), remove culture media and resuspend in 200uL 1xDPBS.
Spin down and remove 1xDPBS (300XG at for 5 mins room temperature).
If using adherent cells: add 50uL of Tryp-LE per well and place plate into a 37C incubator and allow to incubate for 5-10 minutes based on the cell line.
After cells are detached, add 150uL of culture medium to quench the Tryp-LE.
Using a new set of tips for every well and a multichannel pipette, 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 down again at 300xG for 5 minutes, remove media by aspiration.
Add 100uL of cold 1xDPBS with a multichannel to every 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 and remove supernatant.
Resuspend each well in 50uL ice cold OMNI lysis buffer and mix several times with widebore tips.
Add 1uL of distinct hash oligos (10uM) to each well and incubate on ice for 5 mins.
Note: We've found that hashing efficiency can decrease if too little oligos are used. It is best to consider the starting number of cells within each well and target 0.5pmol hash molecules per 1000 cells.
Hashes have the sequence 5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGXXXXXXXXXXBAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3’, where ‘X’s represent the 10nt, well-specific hash ID.
Add 102 uL 1.5% Formaldehyde to each well for FC = 1% and mix with widebore tip.
Allow fixation to occur on ice for 15 minutes.
Hashed nuclei can now be pooled. Combine nuclei (in fixation solution) from all wells into a 15mL conical.
Spin down at 600xG for 10 minutes at 4C.
Resuspend in 1mL of ice cold Nuclei Buffer with 0.1% tween20
Allow to rest on ice for 3 minutes.
Spin down at 600g for 5 minutes at 4C.
Resuspend in 0.5 mL Freezing buffer.
Count cells and bring to final conctration of approx. 5x10e6 cells/mL in freezing buffer (working sln).
Flash freeze and stored at -80C. Otherwise proceed directly to next section.
Note: Freeze thaw may contribute to nuclear lysis, so it is ideal to proceed without freezing when possible; however, the next freeze step is following both ligations and PCR.