TITLE: Overloading and Unpacking (OAK) V1
AUTHORS: Hayley Bennett, Bing Wu

[DESCRIPTION]
High-throughput single-cell sequencing is a powerful technique for investigating the cellular diversity of complex biological systems. Throughput, cost effectiveness, and experimental simplicity are crucial forefronts of technological advancement in profiling single cells. We developed "overloading and unpacking" (OAK), a method that enables robustprofiling of hundreds of thousands of cells in a cost effective manner.

This protocol describes using OAK in conjunction with Chromium Next GEM Single Cell 3ʹ Kit v3.1 reagents.

[STEPS]
SECTION: Before starting
1. Ensure you have adequate aliquots of methanol at -20 °C . We recommend storing in a cool block that can be pulled out and taken to a fume hood for use.

Prepare your single-cell suspension. We recommend starting with over 500K cells if possible, to ensure a visible pellet after fixation.

Pre-cool a centrifuge with a swinging bucket to  4 °C . A swinging bucket is crucial for cell recovery.

Prepare a stock of 3X SSC for washing cells after unpacking.

Prepare twenty labeled 200 µL tubes for aliquoting cells.

Optional: Pre-coat 2 mL microfuge tubes with BSA.
SECTION: Fixation
2. Transfer between 100,000 and 2 million cells to a round-bottom 2 mL microfuge tube in 200 µL PBS.

Slowly add 800 µL ice-cold methanol dropwise to the cells with a P1000, swirling the tube gently.

Add another 800 µL ice-cold methanol in the same manner.

Place cells at  -20 °C  for 15-30min.
Note: longer than 1 hr has been linked to poorer transcript recovery.
SECTION: Post-fix wash
3. Prepare at least 200 µL resuspension buffer and place on ice .

 

Remove cells from -20°C and place on  on ice  for 5 min.

Pellet in a swinging-bucket centrifuge at 1000G for 5 min at 4 °C

Remove as much supernatant as possible without disturbing the pellet and resuspend cells gently in 200 µL resuspension buffer. If cell count is less than 8 million/mL then cells can be pelleted an resuspended again in a smaller volume - this limits the impact of any residual methanol in the cell suspension.
SECTION: Overload 10X Chromium
4. Count the fixed cells and, using the recommendations in the standard 10X Genomics Chromium (CG000204_ChromiumNestGEMSingleCell3_V3.1_Rev_D.pdf) as a guideline, prepare and load the chip with typically 10 times as many cells.

For example, with a solution of 10 million cells/mL, to target 100,000 cells, use 16.5 µL of cell solution and 22.6 µL of nuclease-free water in the reverse transcription mix.
SECTION: Modified reverse transcription
5. After GEM generation transfer the emulsion to a microfuge tube as described in the standard 10X protocol.

Place the reaction in a thermocylcer at 53 °C for 45 min then down to 4 °C. 
Note: Do not include 85°C step, as this will lyse the cells.

Proceed immediately to the next step.
SECTION: Unpacking the GEMS
6. Add 125 µL 10X Genomics recovery agent to the GEMS and allow separation to occur. Do not invert or remove the recovery agent.

Using a low-retention P200 pipette tip, carefully transfer approx 80 µL aqueous solution into a 2 mL microfuge tube.

Add 800 µL of 3X SSC to the cells gently, without pipette mixing.

Proceed immediately to the next step.
SECTION: Washing the cells
7. Pellet cells in a swinging-bucket centrifuge at 650G for 5 min at 4 °C. 
Remove supernatant, being careful not to disturb the pellet.
Add 1 mL of 3X SSC to the cells gently, without pipette mixing.
Pellet cells in a swinging-bucket centrifuge at 650G for 5 min at 4 °C.
SECTION: Distribute the cells into aliquots
8. Work quickly for the best cell recovery.

Remove supernatant, being careful not to disturb the pellet.
Using a low-retention P200 pipette tip add 215 µL to the cell pellet, pipette up and down twice gently to resuspend, then with the same tip, aliquot 10 µL cell solution into twenty different 200 µL microfuge tubes. 

Note: the number of cell aliquots is flexible, however we recommend for loads of 100,000-200,000 cells twenty to reduced the collision rate of indexing.

Immediately store the cells at  -80 °C. 
SECTION: QC of cell yield
9. Using any spare volume of cells that were not aliquoted, at trypan blue and count on a haemocytometer. This will give an expected number of cells per aliquot, this information is useful to allow you to sequence sub-libraries at the correct depth.

Note: some debris is expected, a large amount of debris may indicate some lysis of cells in the experiment and could be detrimental to data quality.
SECTION: Clean up of cell aliquots
10. Heat aliquots to  -85 °C for 5 min.
Clean up using the Dynabeads Silane Viral NA kit (ThermoFisher, 37011D), following the protocol.
Elute cDNA in 35 µL by heating in a thermocycler to 50 °C for 10 min, tapping the tube at the midway point.
Place on a magnet and transfer the eluted cDNA to a new 200 µL microfuge tube. 
SECTION: Amplification of cDNA adding 2nd index
11. This amplification uses a primer against the TSO sequence, and a long P5 primer that adds an index to the barcoded cDNA.

 

For the number of cycles, use that recommended by the standard 10X Genomics Chromium protocol plus two.
 
Store at  4 °C  for up to 72hr or -20 °C for up to a week.
SECTION: Clean up amplified cDNA
12. SPRIselect and QC is performed on the amplified cDNA as described in the standard 10X Genomics Chromium protocol.

Store at  4 °C  for up to 72hr or -20 °C for up to four weeks.
SECTION: Library preparation
13. Fragmentation, End Repair & A-tailing and adaptor ligation steps with clean-up are all perfofmed on the amplified cDNA as described in the standard 10X Genomics Chromium protocol.

At the PCR stage, since we have already added the P5 index earlier, we use a primer to retain this index and add an i7 index.

 
For the number of cycles, use that recommended by the standard 10X Genomics Chromium protocol based on cDNA input.
 
Perform double-sided clean-up as described in the standard 10X Genomics Chromium protocol.

Take 3 µL for QC and store the library at  4 °C  for up to 72hr or -20 °C until sequencing.

Use Qubit for quantification and Tapestation D1000 for size estimation.

Sequence to recommended depth based on the number of cells estimated from the aliquot cell count in Step 9.