Cell harvest, nuclei isolation and sample hashing

Day 0 – (1 hour per cell line)

1. Trypsinize and harvest cell line for plating. 
2. Plating requires 25K cells/well and about 2.5 Million cells/96 well plate.
3. Dilute cells to 250K cells/mL with 10mLs of diluted cells/plate.
4. Using a multichannel pipette and a trough containing diluted cells, aliquot 100uL of cells/well. Use V-bottom culture plate for suspension cells and 96 well culture plates for adherent cells.
5. For large sciPlex screen plate 8 plates per replicate (192 drugs @ 4 doses)

Day 1 – (1.5 hours per 8 plates)

1. Thaw pre-aliquoted drugs.
2. Spin down drugs at 4000 rpm for 1 minute to bring down any drugs on seal
3. Transfer drug plates into the tissue culture hood.
4. Open seal of drug plate and resuspend using a multichannel 10uL pipette with the volume set to 10 uL. After 5 strokes, drugs should be well suspended.
5. Set volume to 1uL and add drug to each culture well.
6. Discard tips and place cells back into the incubator

Day 2 – (3 hours per 8 plates)

Prepare Reagents:

1M Tris HCl ph 7.4

5M  NaCl

1M MgCl2

IGE-PAL

Superase-Inhibitor

Ultrapure BSA

dH2O

20% EM Grade PFA

• Nuclei Buffer (NB)
  • 10mM Tris HCl ph 7.4
  • 10mM NaCl
  • 3mM MgCl2

• Stamp hash plates – Prepared in advance and stored at -20C
  • Deposit 5.0uL of well-specific hashing oligo at 1uM concentration aliquoted in a 96 well plate in 15uL of nuclei buffer 

• NSB – nuclei suspension buffer
  • 10mM Tris HCl ph 7.4
  • 10mM NaCl
  • 3mM MgCl2
  • 1% (v/v) Superase-In 
  • 1% BSA (v/v) from (20mg/mL stock NEB)

• Fixation buffer (22mL per plate processed)
  • 5% PFA (5mL of 20% PFA)
  • 1.25x PBS (2.5mL of 10x PBS)
  • dH20 (12.5mL of nuclease free dH20)

Worksheets for the preparation of CLB, NSB and PFA fixation solutions:

Cold Lysis Buffer (CLB) –

PFA –

1. For adherent cells – (50 minutes)
   1. Remove cells from incubator and bring to the lab bench.
   2. Remove media by aspiration.
   3. Add 100uL of cold 1xPBS with a multichannel to every well to remove residual media.
   4. Remove cold 1xPBS from wells via aspirating or dumping into a waste container.
   5. Add 50uL of Tryp-LE per well and place plate into a 37C incubator.
   6. Allow to incubate for 10-20 minutes based on the cell line
      1. 15 minutes for A549
      2. 20 minutes for MCF7
   7. After cells are properly detached, add 150uL of 1x DMEM + 10% FBS to quench the reaction.
   8. 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 
   9. Spin for 5 minutes at 500g to pellet cells
2. For suspension cells- (10 minutes)
   1. Cells should already be in 96 well V-bottom plate. 
   2. Spin for 5 minutes at 500g to pellet these cells.
3. At this point both suspension cells and adherent cells can be treated the same.
4. Remove media by aspiration or by dumping into a waster container.
5. Add 100uL of cold 1xPBS with a multichannel to every well to wash off residual media.
6. Spin cells down again at 300g for 5 minutes in the V-bottom plate to pellet cells.
7. Add 35uL of ice-cold CLB to each well of the pre-stamped hash plates. These hash plates contain 15uL of pre-aliquoted hashing oligo at the bottom of each well.
8. (Return to cells) Remove cold 1xPBS from wells via aspirating or dumping into a waste container.
9. Add 50uL of hashed CLB solution to each well of the 96 well V-bottom plate using a narrow bore tip. Make sure to resuspend and mix using 3-5 strokes. Discard tips when finished.
10. Resuspend all cells in a 96 well plate. 
11. Upon completion, using a 12 channel multichannel with wide-bore tips add 200uL of fixation buffer. Mix with 3 strokes to ensure complete permeation of the fixative.
12. Incubate each plate for 15 minutes on ice.
13. Pool cells from a single 96 well plate into a trough and transfer with a 10mL pipette mixture into 1 50mL falcon tube.
14. Spin down cells for 5 minutes at 500g.
15. Make sure 3mL of NSB per plate is prepared
16. Resuspend cell pellet in 1mL of NSB (nuclei buffer + 1% Superase-In + 1% BSA) and pool tubes.
17. Spin down cells for 5 minutes at 500g.
18. Resuspend all 8 tubes (if processing 8 plates) in 1 mL of ice cold NSB. Pool all cells into a single falcon tube. Set aside 15uL of nuclei mixture for counting.
19. Spin down nuclei
20. Resuspend nuclei at 2.5 million nuclei/500uL of NSB.
21. Flash freeze and store samples at -80C for further processing by sci-RNA-seq3. 

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