TITLE: Evercode Fixation v2.0.2
AUTHORS: Elisabeth Rebboah, Parse Biosciences

[DESCRIPTION]
This protocol is designed for fixing single cell or single nucleus suspensions for the Parse Biosciences Evercode WT / WT Mega snRNA-seq, "Split-seq".

[BEFORE_START]
For additional questions not discussed below, please contact us at support@parsebiosciences.com. We also have a library of additional resources and videos on our support site at https://support.parsebiosciences.com/.
Sample Input: This protocol begins with a single cell suspension. If you are fixing cells that were previously frozen, ensure the suspension is thawed before beginning. For cell samples, we recommend suspensions with >70% viability, <5% aggregation/debris, and 100,000 or more cells. For nuclei samples, we recommend suspensions with 100,000 or more nuclei. We recommend minimizing the length of time samples are stored on ice prior to fixation, as it can negatively impact results. If you have questions about your starting material, please contact us at support@parsebiosciences.com.
Cell Detachment: If dissociating adherent cell line samples, we recommend TrypLE Express Enzyme (1X), phenol red (Thermo Fisher Scientific). Due to high RNase activity, we do not recommend dissociation with standard trypsin, which may reduce gene and transcript detection.
Centrifugation: Use a swinging bucket rotor for all high-speed centrifugation steps in this protocol. Use of a fixed-angle rotor will lead to substantial cell/nuclei loss. Although the recommended centrifugation speeds are appropriate for most sample types, they can be adjusted to improve retention. Ideal centrifugation speed and duration should be empirically determined to optimize retention and resuspension efficiencies.
Avoiding RNase Contamination: Standard precautions should be taken to avoid introducing RNases into samples or reagents throughout the workflow. Always wear proper laboratory gloves and use aseptic technique. RNases are not inactivated by ethanol or isopropanol but can be inactivated by products such as RNaseZap RNase Decontamination Solution (Thermo Fisher Scientific). These can be sprayed on benchtops and pipettes. Filtered pipette tips should be used to reduce RNase contamination from pipettes.
Addition of BSA: BSA can prevent aggregation of nuclei and some cell types prone to clumping, including PBMCs. For nuclei, BSA should be added to the Nuclei Buffer as described in the protocol. If your cell type is prone to clumping, we strongly recommend adding BSA to the Cell Prefixation Buffer as described in the protocol. If you have lower cell numbers or you are unsure if your cell type fits this category, we also recommend adding BSA. We strongly recommend using Gibco Bovine Albumin Fraction V (7.5% solution) (Thermo Fisher Scientific), which was chosen based on its very low RNase activity.
Cell Strainers: A 40 μm cell strainer will be used in multiple steps. To maximize cell retention, press the pipette tip directly against the strainer. Ensure that ample pressure is applied to hold contact between the tip and the strainer to force liquid through in ~1 second. For cells larger than 40 μm, the 40 μm strainer should be replaced throughout the protocol with the appropriate size mesh (70 μm or 100 μm).
Reagent Stability: After Cell Fixation Solution Additive or RNase Inhibitor are added to Cell Fixation Solution, Cell Prefixation Buffer, Cell Buffer, and Nuclei Buffer as indicated in the protocol, the mixed reagents are stable for 1 month when stored at -20°C and can be freeze-thawed once. Additional storage or freeze-thaws will compromise data quality.
Cell/Nuclei Counting and Quality Assessment: We recommend a hemocytometer for cell counting, but alternative cell counting devices can also be used. If possible, we recommend validating counts from alternative devices to a hemocytometer when first using Evercode Fixation kits. To assess sample quality, we also recommend use of viability stains like trypan blue or acridine orange and propidium iodide (AOPI). As debris and cell/nuclei clumping can impact counts and can be difficult to assess, the figure below shows some samples of varying quality. When first using Evercode Fixation kits, we suggest saving images from each counting step.
 
Optimizing Cell/Nuclei Recovery: It is critical that cells/nuclei are thoroughly resuspended after centrifugation throughout the protocol. Resuspend by slowly and repeatedly pipetting up and down until no clumps are visible. Due to cell adherence to tubes, we also recommend carefully pipetting up and down along the bottom and sides of tubes to minimize cell loss. Similarly, we do not recommend wide bore pipette tips as they make it difficult to adequately resuspend cell/ nuclei pellets.
Cell/nuclei adherence to plastic can impact cell recovery throughout the protocol and impact sequencing data. Ensure that the 15 mL centrifuge tubes that will be used are polypropylene and not polystyrene. Polystyrene tubes will lead to substantial cell loss. BSA can also prevent cell adhesion to plastics. Thus, we recommend blocking 15 mL polypropylene centrifuge tubes with BSA to increase cell retention, especially for samples with fewer cells. See the Appendix for a blocking protocol.
Ensure the correct cell strainer is used based on the diameter of the cells you are processing (see "Cell Strainers" in Notes Before Starting for more details).
For the first few times you use Evercode Fixation kits, we recommend retaining the supernatants removed in steps 1.2.5 and 1.2.13 (for cell fixation) or 2.2.5 and 2.2.13 (for nuclei fixation). In the unlikely event of very high sample loss, these supernatants can be analyzed to identify points for optimization.

[STEPS]
SECTION: Section 1: Cell fixation
1. Cell Fixation Setup
This protocol is designed for fixing single cell suspensions which will be prepared in step 1.2.1. 

Note: If you are fixing nuclei, proceed to the Nuclei Fixation Protocol.
SECTION: Section 1: Cell fixation
1.1. (Optional) To maximize cell retention, prepare two BSA coated 15 mL centrifuge tubes per sample being fixed, according to the protocol in the Appendix.
SECTION: Section 1: Cell fixation
1.2. (Optional) If you do not plan to immediately process samples with an Evercode Whole Transcriptome kit after fixation, place a Mr. Frosty Freezing Container (or equivalent) at room temperature.
SECTION: Section 1: Cell fixation
1.3. Cool the centrifuge with swinging bucket rotor to 4C.
SECTION: Section 1: Cell fixation
1.4. Fill a bucket with ice and proceed to the next step.
SECTION: Section 1: Cell fixation
1.5. Gather the following items and handle as indicated below. It is important that all solutions (except DMSO) are kept on ice after thawing.

 
Critical! All items should be fully thawed before moving to the next step. Ensure that DMSO is not stored on ice.
SECTION: Section 1: Cell fixation
1.6. If using this set of reagents for the first time, proceed to step 1.1.7. Otherwise, check the date on the Cell Fixation Reagents kit box. If less than 1 month has elapsed, proceed to step 1.1.11.
Note: Evercode Cell Fixation kits previously mixed by the user should have a date on the Cell Fixation Reagents kit box and a mark on the caps of the Cell  Fixation Solution, Cell Fixation Buffer, and Cell Buffer tubes. After mixing reagents, Evercode Cell Fixation kits should only be freeze-thawed once and stored for up to 1 month at -20C. Longer storage or additional freeze-thaws will compromise data quality.
SECTION: Section 1: Cell fixation
1.7. Add 550 μL of Cell Fixation Additive directly into the Cell Fixation Solution. Mix thoroughly by pipetting up and down 5x with a P1000 set to 750 μL.

To record the addition of Cell Fixation Additive, mark the cap of the Cell Fixation Solution tube, and store on ice.

SECTION: Section 1: Cell fixation
1.8. Add 50 μL of RNase Inhibitor directly into the Cell Prefixation Buffer tube. Mix thoroughly by pipetting up and down 5x with a P1000 set to 750 μL.

To record the addition of RNase Inhibitor, mark the cap of the Cell Prefixation Buffer tube, and store on ice.
SECTION: Section 1: Cell fixation
1.9. Add 17 μL of RNase Inhibitor directly into the Cell Buffer tube. Mix thoroughly by pipetting up and down 5x with a P1000 set to 750 μL.

To record the addition of RNase Inhibitor, mark the cap of the Cell Buffer tube, and store on ice.
SECTION: Section 1: Cell fixation
1.10. Record today's date on the Cell Fixation Reagents kit box.
Note: After mixing reagents, Evercode Cell Fixation kits should only be freeze-thawed once and stored for up to 1 month at -20C. Longer storage or additional freeze-thaws will compromise data quality.
SECTION: Section 1: Cell fixation
1.11. (Optional) If your sample is cell-limited or prone to clumping (such as PBMCs), it is recommended to add 7.5% Gibco BSA Fraction V to the Cell Prefixation Buffer. For each sample being fixed, prepare Cell Prefixation Buffer + BSA according to the table below. Cell Prefixation Buffer + BSA should be prepared fresh and used the same day. Mix thoroughly by pipetting up and down 5x and store on ice.

 
Critical! Ensure the Cell Prefixation Buffer contains RNase Inhibitor, as marker on the tube cap.
SECTION: Section 1: Cell fixation
2. Cell Fixation Protocol
Section 1.1 should have been completed before proceeding. Ensure tube caps have been marked when reagents were mixed and no more than 1 month has elapsed since the time of mixing, as dated on the Cell Fixation Reagents kit box.
SECTION: Section 1: Cell fixation
2.1. Create a single cell suspension for the samples you plan to fix and store them on ice. When possible, avoid prolonged incubation on ice prior to fixation.
SECTION: Section 1: Cell fixation
2.2. Count the number of cells in your sample with a hemocytometer or alternative counting device and record the count. Keep cells on ice during counting and work quickly to minimize time on ice prior to fixation.
SECTION: Section 1: Cell fixation
2.3. Transfer up to 4 million cells into a 15 mL polypropylene centrifuge tube and store on ice.
Critical!  No more than 4 million cells should be used for any single sample. Exceeding this number may result in substantially elevated doublet rates. The minimum recommended number of cells to proceed with is 100,000. It is possible to be successful with fewer cells, but it is not recommended as pelleting cells becomes more difficult.
SECTION: Section 1: Cell fixation
2.4. Centrifuge the 15 mL tube in a swinging bucket rotor for 10 minutes at 200 x g at 4°C.
SECTION: Section 1: Cell fixation
2.5. Remove and discard the supernatant. Fully resuspend the pellet in 750 μL of cold Cell Prefixation Buffer or (if prepared in step 1.1.11) Cell Prefixation Buffer + BSA with a P1000 set to 750 μL.

Critical! Failure to fully resuspend cells may result in substantially elevated doublet rates. For this reason, do NOT use a wide bore pipette tip as it makes it difficult to fully resuspend cells.
SECTION: Section 1: Cell fixation
2.6. Pipette cells through a 40 μm strainer into a new 15 mL polypropylene centrifuge tube with a P1000 and store on ice.

Note: For cells larger than 40 μm, the 40 μm strainer should be replaced throughout the protocol with the appropriate size mesh (70 μm or 100 μm).

Critical! To ensure that all of the liquid passes through the strainer, press the tip of the pipette against the filter and steadily depress down the pipette plunger. All of the liquid should pass through the strainer in ~1 second.
SECTION: Section 1: Cell fixation
2.7. Add 250 μL of Cell Fixation Solution to the 15 mL tube and mix immediately by pipetting up and down exactly 3x with a P1000 set to 250 μL. Return the tube to ice.

Critical! Do NOT perform additional mixing at this step. Also, ensure the Cell Fixation Solution contains Cell Fixation Additive, as indicated by a mark on the tube cap.
SECTION: Section 1: Cell fixation
2.8. Incubate on ice for 10 minutes.
SECTION: Section 1: Cell fixation
2.9. Add 80 μL of Cell Permeabilization Solution to the 15 mL tube and mix thoroughly by pipetting up and down 3x with a P1000 set to 250 μL. Return the tube to ice.
SECTION: Section 1: Cell fixation
2.10. Incubate on ice for 3 minutes.

Note: Do NOT vortex the Cell Neutralization Buffer. Prior to use, invert the tube to mix.
SECTION: Section 1: Cell fixation
2.11. Add 4 mL of Cell Neutralization Buffer to the 15 mL tubes. Gently invert the 15 mL tube once to mix and return to ice.
SECTION: Section 1: Cell fixation
2.12. Centrifuge the 15 mL tube in a swinging bucket rotor for 10 minutes at 200 x g at 4°C.
SECTION: Section 1: Cell fixation
2.13. Remove and discard the supernatant. Fully resuspend each pellet in 150 μL of cold Cell Buffer with a P1000 set to 150 μL and return to ice.
SECTION: Section 1: Cell fixation
2.14. Pipette cells through a 40 μm strainer into a new 1.5 mL tube with a P1000 and store on ice.
SECTION: Section 1: Cell fixation
2.15. If immediately processing samples with an Evercode Whole Transcriptome kit, proceed to the appropriate user guide. Otherwise, proceed to step 1.2.16.
SECTION: Section 1: Cell fixation
2.16. Add 2.5 μL of DMSO. Gently flick the tube 3x to mix.
SECTION: Section 1: Cell fixation
2.17. Incubate on ice for 1 minute.
SECTION: Section 1: Cell fixation
2.18. Repeat steps 16 and 17 two more times for a total addition of 7.5 μL of DMSO.
SECTION: Section 1: Cell fixation
2.19. Mix gently by pipetting up and down 5x with a P200 set to 75 μL. Avoid creating bubbles.

Critical! Do NOT vortex cells.
SECTION: Section 1: Cell fixation
2.20. Count the number of cells in your sample with a hemocytometer or alternative counting device and record the count. Keep cells on ice during counting and work quickly to minimize the time that fixed cells are out.
SECTION: Section 1: Cell fixation
2.21. (Optional) If your sample has more than 500,000 cells, we recommend splitting it into two
1.5 mL tubes prior to storage.
SECTION: Section 1: Cell fixation
2.22. Store tubes in a Mr. Frosty Freezing Container (or equivalent device) at -80°C, according to the manufacturer’s instructions.

Note: Storing samples directly in the freezer without controlled cooling may lead to cell damage and compromise data quality.
SECTION: Section 2: Nuclei Fixation
3. Nuclei Fixation Setup
This protocol is designed for fixing single nuclei suspensions which will be prepared in step 2.2.1. 

Note: If you are fixing cells, refer back to the Cell Fixation Protocol.
SECTION: Section 2: Nuclei Fixation
3.1. (Optional) To maximize cell retention, prepare two BSA coated 15 mL centrifuge tubes per sample being fixed, according to the protocol in the Appendix.

Note: Although step 1 is options, 7.5% Gibco BSA Fraction V is required for other parts of the protocol.
SECTION: Section 2: Nuclei Fixation
3.2. (Optional) If you do not plan to immediately process samples with an Evercode Whole Transcriptome kit after fixation, place a Mr. Frosty Freezing Container (or equivalent) at room temperature.
SECTION: Section 2: Nuclei Fixation
3.3. Cool the centrifuge with swinging bucket rotor to 4C.
SECTION: Section 2: Nuclei Fixation
3.4. Fill a bucket with ice and proceed to the next step.
SECTION: Section 2: Nuclei Fixation
3.5. Gather the following items and handle as indicated below. It is important that all solutions (except DMSO) are kept on ice after thawing.

 

Critical! All items should be fully thawed before moving to the next step. Ensure that DMSO is not stored on ice.
SECTION: Section 2: Nuclei Fixation
3.6. If using this set of reagents for the first time, proceed to step 2.1.7. Otherwise, check the date on the Nuclei Fixation Reagents kit box. If less than 1 month has elapsed, proceed to step 2.1.8.

Note: Evercode Nuclei Fixation kits previously mixed by the user should have a date on
the Nuclei Fixation Reagents kit box and a mark on the of the Nuclei Buffer tube. After
mixing reagents, Evercode Nuclei Fixation kits should only be freeze-thawed once and
stored for up to 1 month at -20°C. Longer storage or additional freeze-thaws will
compromise data quality.
SECTION: Section 2: Nuclei Fixation
3.7. Add 63 μL of RNase Inhibitor directly into the Nuclei Buffer tube. Mix thoroughly by pipetting up and down 5x with a P1000 set to 750 μL.

To record the addition of RNase Inhibitor, mark the cap of the Nuclei Buffer tube and store on ice.

Record today’s date on the Nuclei Fixation Reagents kit box.

Note: After mixing reagents, Evercode Nuclei Fixation kits should only be freeze-thawed
once and stored for up to 1 month at -20°C. Longer storage or additional freeze-thaws will compromise data quality.
SECTION: Section 2: Nuclei Fixation
3.8. For each sample being fixed, prepare Nuclei Buffer + BSA in a new tube. You will need Nuclei Buffer (without BSA) for later use. Nuclei Buffer + BSA should be prepared fresh and used the same day. Mix by pipetting up and down 5x with a P1000 set to 750 μL and store both Nuclei Buffer + BSA and Nuclei Buffer (without BSA) on ice.

 
Critical! Ensure the Nuclei Buffer contains RNase Inhibitor, as marker on the tube cap.
SECTION: Section 2: Nuclei Fixation
4. Nuclei Fixation Protocol
Section 2.1 should have been completed before proceeding. Ensure tube caps have been marked when reagents were mixed and no more than 1 month has elapsed since the time of mixing, as dated on the Nuclei Fixation Reagents kit box.
SECTION: Section 2: Nuclei Fixation
4.1. Create a single nuclei suspension for the samples you plan to fix and store them on ice. When possible, avoid prolonged incubation on ice prior to fixation.
SECTION: Section 2: Nuclei Fixation
4.2. Count the number of nuclei in your sample with a hemocytometer or alternative counting device and record the count. Keep nuclei on ice during counting and work quickly to minimize time on ice prior to fixation.
SECTION: Section 2: Nuclei Fixation
4.3. Transfer up to 4 million nuclei into a 15 mL polypropylene centrifuge tube and store on ice.
Critical! No more than 4 million nuclei should be used for any single sample. Exceeding this number may result in substantially elevated doublet rates. The minimum recommended number of nuclei to proceed with is 100,000. It is possible to be successful with fewer nuclei, but it is not recommended as pelleting nuclei becomes more difficult.
SECTION: Section 2: Nuclei Fixation
4.4. Centrifuge the 15 mL tube in a swinging bucket rotor for 10 minutes at 200 x g at 4°C.
SECTION: Section 2: Nuclei Fixation
4.5. Remove and discard the supernatant. Fully resuspend the pellet in 750 μL of cold Nuclei Buffer + BSA with a P1000 set to 750 μL.

Critical! Failure to fully resuspend nuclei may result in substantially elevated doublet rates. For this reason, do NOT use a wide bore pipette tip as it makes it difficult to fully resuspend nuclei.
SECTION: Section 2: Nuclei Fixation
4.6. Pipette nuclei through a 40 μm strainer into a new 15 mL polypropylene centrifuge tube with a P1000 and store on ice.

Critical!  To ensure that all of the liquid passes through the strainer, press the tip of the pipette against the filter and steadily depress down the pipette plunger. All of the liquid should pass through the strainer in ~1 second.
SECTION: Section 2: Nuclei Fixation
4.7. Add 250 μL of Nuclei Fixation Solution to the 15 mL tube and mix immediately by pipetting up and down exactly 3x with a P1000 set to 250 μL. Return the tube to ice.

Critical!  Do NOT perform additional mixing at this step.
SECTION: Section 2: Nuclei Fixation
4.8. Incubate on ice for 10 minutes.
SECTION: Section 2: Nuclei Fixation
4.9. Add 80 μL of Nuclei Permeabilization Solution to the 15 mL tube and mix thoroughly by pipetting up and down 3x with a P1000 set to 250 μL. Return the tube to ice.
SECTION: Section 2: Nuclei Fixation
4.10. Incubate on ice for 3 minutes.

Note: Do NOT vortex the Nuclei Neutralization Buffer. Prior to use, invert the tube 5x to mix.
SECTION: Section 2: Nuclei Fixation
4.11. Add 4 mL of Nuclei Neutralization Buffer to the 15 mL tube. Gently invert the 15 mL tube once to mix and return to ice.
SECTION: Section 2: Nuclei Fixation
4.12. Centrifuge the 15 mL tube in a swinging bucket rotor for 10 minutes at 200 x g at 4°C.
SECTION: Section 2: Nuclei Fixation
4.13. Remove and discard the supernatant. Fully resuspend the pellet in 150 μL of cold Nuclei Buffer (without BSA but with RNase Inhibitor added) with a P1000 set to 150 μL and return to ice.
SECTION: Section 2: Nuclei Fixation
4.14. Pipette nuclei through a 40 μm strainer into a new 1.5 mL tube with a P1000 and store on
ice.
SECTION: Section 2: Nuclei Fixation
4.15. If immediately processing samples with an Evercode Whole Transcriptome kit, proceed to the appropriate user guide. Otherwise, proceed to step 2.2.16.
SECTION: Section 2: Nuclei Fixation
4.16. Add 2.5 μL of DMSO. Gently flick the tube 3x to mix.
SECTION: Section 2: Nuclei Fixation
4.17. Incubate on ice for 1 minute.
SECTION: Section 2: Nuclei Fixation
4.18. Repeat steps 16 and 17 two more times for a total addition of 7.5 μL of DMSO.
SECTION: Section 2: Nuclei Fixation
4.19. Mix gently by pipetting up and down 5x with a P200 set to 75 μL. Avoid creating bubbles.
Critical! Do NOT vortex nuclei.
SECTION: Section 2: Nuclei Fixation
4.20. Count the number of nuclei in your sample with a hemocytometer or alternative counting device and record the count. Keep nuclei on ice during counting and work quickly to minimize the time that fixed nuclei are out.
SECTION: Section 2: Nuclei Fixation
4.21. (Optional) If your sample has more than 500,000 nuclei, we recommend splitting it into two 1.5 mL tubes prior to storage.
SECTION: Section 2: Nuclei Fixation
4.22. Store tubes in a Mr. Frosty Freezing Container (or equivalent device) at -80°C, according to the manufacturer’s instructions.
Note: Storing samples directly in the freezer without controlled cooling may lead to nuclei
damage and compromise data quality.
SECTION: Appendix
5. Appendix: Tube Blocking with BSA
Blocking the 15 mL polypropylene centrifuge tubes used in the Cell or Nuclei Fixation Protocols with BSA can increase cell/nuclei yield. This is especially helpful for cells prone to sticking to plastic or when working with low cell/nuclei counts.
SECTION: Appendix
5.1. Prepare a fresh 1% BSA Master Mix as follows, depending on the number of tubes you want to block.

 

Note: Two 15 mL polypropylene centrifuge tubes are needed for each sample.
SECTION: Appendix
5.2. Fill each 15 mL tube with the 1% BSA Master Mix and cap the tubes.
SECTION: Appendix
5.3. Incubate the tubes for 30 minutes at room temperature.
SECTION: Appendix
5.4. Decant and discard the 1% BSA Master Mix. Remove any remaining solution from the bottom of the tube with a P1000.
SECTION: Appendix
5.5. With the caps removed, incubate the tubes for 30 minutes in a biosafety cabinet at room temperature.
SECTION: Appendix
5.6. Proceed to the appropriate Cell or Nuclei Fixation Protocol, or store BSA coated tubes at 4°C for up to 4 weeks.