key: cord-0070064-2uzqn5wn authors: Genge, Palak C.; Roll, Charles R.; Heubeck, Alexander T.; Swanson, Elliott; Kondza, Nina; Lord, Cara; Weiss, Morgan; Hernandez, Veronica; Phalen, Cole; Thomson, Zachary; Torgerson, Troy R.; Skene, Peter J.; Bumol, Thomas F.; Reading, Julian title: Optimized workflow for human PBMC multiomic immunosurveillance studies date: 2021-11-05 journal: STAR Protoc DOI: 10.1016/j.xpro.2021.100900 sha: a548d845f0857ef33e042aeaf7698f27b59ce6a6 doc_id: 70064 cord_uid: 2uzqn5wn Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021). Deep immune profiling is essential for understanding the human immune system in health and disease. Successful biological interpretation of this data requires consistent laboratory processing with minimal batch-to-batch variation. Here, we detail a robust pipeline for the profiling of human peripheral blood mononuclear cells by both high-dimensional flow cytometry and single-cell RNA-seq. These protocols reduce batch effects, generate reproducible data, and increase throughput. For complete details on the use and execution of this protocol, please refer to Savage et al. (2021) . Prepare workspace This protocol should be performed in a Biosafety level 2 laboratory. All cell processing including 10x Genomics GEM Generation should be performed in a Class II Biological Safety Cabinet (BSC) with a vacuum connection for aspirating media. The vacuum line must be connected to a vacuum trap containing bleach. Ensure that the BSC, pipettes, and additional equipment to be used in the BSC such as the Integra VIAFLO 96 and 103 Chromium Controllers are either sterile or have been cleaned with 70% Reagent Alcohol prior to use. Turn on centrifuges and chill to 4C. After the 10x GEM-generation step has been completed, the protocol can continue in a PCR-Free working area to avoid potential contamination from unrelated amplicons. After the cDNA amplification step has been completed, libraries can be prepared in a ''post-amplification'' working area. Regulatory compliance for use of primary human material Peripheral blood mononuclear cells (PBMC) used in this study were collected with written informed consent under the supervision of an appropriate Institutional Review Board (IRB) Protocol. Human samples were de-identified and assigned bar code labels linking to metadata such as subject number, blood draw type etc. For longitudinal studies where multiple assay batches will be run and compared, a consistent PBMC batch control sample should be included to assess the quality of each assay run and to provide a consistent control for batch-to-batch correction of data. In our case, a single donor leukopak, Quant-iTä In this section, the procedure for thawing human PBMC for subsequent High Dimensional Flow Cytometry and 10x Genomics v3.1 scRNA-seq (single-cell RNA-sequencing) with antibody-based cell hashing workflows is described. Typically, a batch of 23 PBMC samples and one bridging control leukopak sample are processed for downstream assays. Adding thawed PBMC directly to pre-warmed media streamlines the first step of the thaw, and the use of AIM V media eliminates serum (a potential source of activating compounds as well as lot-to-lot variability). Note: If working with fresh or cultured cells, proceed to ''cell count and normalization''. 1. Prepare cell thawing media a. Transfer 30 mL of AIM V media into one 50 mL conical tube for each sample to be processed. Place on ice until ready to thaw cells. b. Transfer 40 mL of AIM V media into one 50 mL conical tube for each sample to be processed. 30 min prior to cell thawing, place the 40 mL AIM V tubes into a 37 C water bath. Note: Other serum-free media may be used in place of AIM V. Timing: 30 min The purpose of this step is to quantify the cell concentration of each sample, to resuspend each sample at the appropriate concentration for downstream assays. The Nexcelom Cellaca MX highthroughput cell counter performs 24 cell counts in three minutes. The fluorescent AOPI (Acridine orange/Propidium iodide) method quantifies viable nucleated cells (AO-positive green cells) and dead nucleated cells (PI-positive red cells). As PI quenches AO, a viable cell count and dead cell count are generated. As only nucleated cells are counted, contaminating red blood cells are excluded from the count, eliminating the need for red blood cell lysis. The thawed PBMC samples, normalized to 10 million viable cells per mL in ice cold DPBS, are compatible with both our 10x Genomics scRNA-seq and high dimensional flow cytometry workflows. Note: Alternative cell counting methods such as Trypan Blue dye exclusion plus red blood cell lysis may be used in place of high-throughput AOPI cell counting. For small batches of samples, a manual hemacytometer may also be suitable. 6. Load Cellaca MX cell counter plate (Figure 1 GEMs then undergo reverse transcription (GEM-RT) after which can be stored at 2 C-8 C for up to 72 h or À20 C for up to a week. 10. Cell Hashing a. Make Human TruStain FcX and 10% BSA Master Mix for the appropriate number of reactions. b. Transfer 500,000 Human PBMCs in 50 mL DPBS from the deep well masterplate (see Cell count and normalization section) to sample plate. c. Add 18.7 mL of the Human TruStain FcX and 10% BSA Master Mix to each sample. d. Set multi-channel pipette to 60 mL and slowly pipette-mix 5 times. e. Apply Microseal 'B' adhesive seal and incubate on ice for 10 min. f. Prepare a master mix of 1 mL (0.5 mg) of each unique TotalSeqä-A anti-human Hashtag Antibody and 31.3 mL 2% BSA/DPBS (RNA-seq Staining Buffer) for each sample. Include a 20% overage or slop volume. g. Set multi-channel pipette to 80 mL and slowly pipette-mix 5 times. h. Apply adhesive seal and incubate plate on ice for 20 min. i. During the incubation step turn on Integra VIAFLO 96 and click ''RUN'' to home instrument. Follow prompt on screen for further set-up. j. Prepare Integra platform, pipette tips (7 per sample), reservoirs, reservoir tray, and Integra cold block ( Figure 3 ). CRITICAL: Set up Integra platform and save operation programs in VIAFLO 96 before starting. CRITICAL: When running the Integra VIAFLO 96 programs ensure that the platform is set such that the plate in ''B'' aligns with the ''B'' position on the instrument and the black adjustment lever is in the middle/straight position to ensure that the tips, reservoir, and plate align during the automated wash steps. CRITICAL: Use new pipette tips for every removal and resuspension step (7 tips per sample). CRITICAL: During the following ''Wash'' thaw 10x GEM Generation reagents as described in step 3a. k. Add 60 mL of 2% BSA/DPBS to an Integra reservoir and hold on ice when not in use. l. Set the reservoir containing 60 mL of 2% BSA/DPBS tray and place it in the ''AB'' position on the Integra VIAFLO 96 stage. m. Remove the seal from the sample plate and place the sample plate in an Integra cold block on the instrument in the ''B'' position. n. Run the 'RNA-Seq Wash 1 0 program to add 150 mL 2% BSA/DPBS solution to each sample and mix 5 times. o. Transfer the plate in a cold-block to a stable surface. Seal plate with a Micro-Optical qPCR adhesive seal, and centrifuge at 750 g for 5 min at 2 C-8 C (ensure centrifuge is pre-cooled). During each centrifugation step, hold the cold block on ice. p. Carefully transfer the pelleted sample plate to the cold block and back to the ''B'' position on the Integra VIAFLO 96. Remove the seal. q. Replace the buffer reservoir with a new empty reservoir and label it ''Waste.'' r. Run the 'RNA-Seq Remove' program on Integra to remove 200 mL of the clear supernatant from each well, leaving 50 mL above the pellet. s. With the buffer reservoir in place, run the 'RNA-Seq Wash 2 0 program on the Integra. t. Transfer plate in cold block to a stable surface, seal plate with a Micro-Optical qPCR adhesive seal, and centrifuge at 750 g for 5 min at 2 C-8 C (ensure centrifuge is pre-cooled). u. Carefully transfer the pelleted sample plate to the cold block and back to the ''B'' position on the Integra VIAFLO 96. Remove the seal. v. With the waste reservoir in place, run the 'RNA-Seq Remove' program on Integra. w. With the buffer reservoir in place, run the 'RNA-Seq Wash 2 0 program. x. Transfer plate to a stable surface, seal plate with a Micro-Optical qPCR adhesive seal, and centrifuge at 750 g for 5 min at 2 C-8 C (ensure centrifuge is pre-cooled). y. With the waste reservoir in place, run the 'RNA-Seq Remove' program on Integra. z. With the buffer reservoir in place, run the 'RNA-Seq Final' program on the Integra to resuspend the cell pellet. Total volume is now 200 mL. aa. Transfer the plate to ice and proceed immediately to the next step. a. Refer to ''Cell count and normalization'' section to count samples on the Cellaca MX cell counter. Use 27.5 mL of sample and 27.5 mL of AOPI dye to conserve sample volume. CRITICAL: Due to high background noise from oligo-conjugated, cell hashing antibodies use the MX04.0_AOPI_scRNA-Pipeline Assay custom program (Fluorescence exposure settings F1, 300 msec and F2, 350 msec). b. Use the live cell concentration to normalize pools such that each sample is pooled at an equal fraction and will be sufficient to overload at up to 64K cells per well during 10x GEM generation. CRITICAL: A cell viability cut-off of 70% is recommended to ensure the quality of the data, however differences in samples due to a patient's disease profile should be considered. In past preparations, cell viabilities as low as 50% have been processed due to patient disease profile. See troubleshooting section problem 2 for potential solution if cell viability is low. c. Based on the number of 103 wells to be run, calculate the amount of batch control to spike into each pool (should be 1/5th of the amount of a sample i.e., for 12 samples pooled at 100K cells/sample, pool 20K cells of the bridging control). d. Pass the pool through a labeled 35 mM cell strainer cap securely held in place on top of a 1.5 mL screw-top centrifuge tube. e. Take a cell count of the pool using the Nexcelom Cellometer. Mix 12.5 mL of AOPI + 12.5 mL sample pool, pipette-mix, remove plastic from Cellometer slide and place on a clean surface, load 20 mL of AOPI stained sample pool to the slide chamber. Record the total cells/mL and viability. f. Dilute the sample pool in DPBS for 103 GEM Generation. Use the following equation to determine volume (mL) of the sample pool must be diluted in DPBS for 103 GEM Generation: d. Vortex the Gel Beads for 30 s, remove strip tube from holder and briefly centrifuge, and return to holder. e. Slowly aspirate 50 mL Gel Beads. Dispense into row 2 without introducing bubbles. Wait 30 s before moving to the next step. f. Dispense 45 mL Partitioning Oil into row 3 wells. g. Attach the 10x Gasket and ensure the gasket holes are aligned with the wells. Avoid touching the smooth surface ( Figure 5 ). h. Load the chip into the 10x Chromium controller and press play. Avoid tilting the chip in the process. ($18 min run time) (Figure 6 ). i. IMMEDIATELY unload the chip, discard the gasket, and fold the lid back until the holder clicks into place so the chip is sitting at a 45 degree angle (Figure 7 ). j. Slowly aspirate 100 mL GEMs from the lowest point of row 3 slowly rotating the pipette away to ensure that the tips stay at the lowest point (Don't allow a seal to form between the tips and the well) ( Figure 8 ). k. Taking $20 s, dispense GEMs into a semi-skirted 96-well PCR plate on ice, keeping pipette tips against the sidewalls of the wells at a 45 degree angle. l. Repeat for each additional chip as needed. Keep GEMs on ice no longer than an hour. m. Load the plate onto a Thermal Cycler and run the 'GEM RT 0 program with the following cycling conditions Pause point: store at 2 C-8 C for up to 72 h or À20 C for up to one week. GEM recovery, cDNA amplification, HTO library preparation i. If biphasic separation is incomplete, securely seal the plate with a Micro-Optical qPCR adhesive seal, invert 5 times and briefly centrifuge. f. Pipetting from the bottom of the well, slowly remove and discard 125 mL of recovery agent (pink) without removing any clear or opaque sample ( Figure 9 ). g. Vortex the Cleanup Mix well and add 200 mL to each sample. Heat-seal the plate and invert 103 to mix. h. Incubate for 10 min at 20 C-25 C. i. At the 5-min mark, invert 103 to mix again. Incubate at 20 C-25 C for 2 min. Optional: Seal and briefly centrifuge when incubation is complete. q. Place the plate on a magnetic plate stand, once the solution clears, transfer 35 mL of sample to a new plate. r. Make up the cDNA Amplification Reaction Mix. s. Add 65 mL cDNA Amplification Reaction Mix to each 35 mL sample well. t. Heat seal, cool immediately, invert 5x and centrifuge. u. Run the following ''cDNA Amplification'' program with 11 cycles of amplification Pause point: store at 4 C for up to 72 h or À20 C for up to one week. v. Add 60 mL SPRI beads to each sample well, incubate for 5 min at 20 C-25 C. w. Place on a magnetic plate stand to pellet beads for 10 min. Transfer 155 mL of the clear supernatant to a new semi-skirted 96-well PCR plate. x. Heat seal the ''HTO'' plate and store it on ice while you complete the following steps on the gene expression libraries. i. Add 100 mL SPRI beads directly to the resuspended beads. Incubate for 10 min at 20 C-25 C. j. Place on a magnetic plate stand for 10 min and discard the clear supernatant. k. Add 200 mL 80% EtOH to the pellet. Wait 30 s. l. Remove and discard EtOH, being careful to not touch the beads. m. Repeat steps k and l for a total of 2 washes. u. Add 160 mL SPRI beads, heat seal, vortex to mix. Incubate at 20 C-25 C for 5 min. v. Place on magnetic plate stand and discard the clear supernatant w. Add 200 mL 80% EtOH to each well avoiding the pellet, wait 30 s. x. Remove and discard EtOH. y. Repeat for a total of 2 washes. z. Centrifuge briefly and remove remaining EtOH. Air dry on a magnetic plate stand for 30 s. aa. Resuspend the beads in 30 mL Elution Buffer, incubate for 5 min at 20 C-25 C. bb. Place on a magnetic plate stand and transfer 30 mL of sample to a new plate. cc. Make 1:10 dilution (45 mL Elution Buffer + 5 mL HTO Final Libraries) and record the barcode and well location. dd. QC HTO Libraries alongside the RNA once library prep is complete. 16. Gene Expression Library Intermediate QC a. Run 1 mL of the 1:10 diluted Gene Expression Library prior to continuing library preparation on an Agilent Bioanalyzer or Fragment Analyzer to report cDNA quality and yield. Timing: 3.5 h Transfer to a pre-cooled thermal cycler and run the following ''Fragmentation and A-Tail'' program. g. Add 30 mL SPRI beads to each sample well, heat seal, cool, and vortex. Incubate for 5 min at 20 C-25 C. h. Place the plate on a magnetic plate stand for at least 3 min and transfer 75 mL of clear supernatant to a new set of columns in the plate. i. Add 10 mL SPRI beads to each sample heat seal, cool, and vortex. Incubate for 5 min at 20 C-25 C. j. Place on a magnetic plate stand and discard the clear supernatant. k. Add 125 mL 80% EtOH to each well avoiding the pellet, wait 30 s. l. Remove and discard EtOH. m. Repeat steps k and l for a total of 2 washes. n. Centrifuge briefly, place plate back on the magnet and remove remaining EtOH. Air dry on a magnetic plate stand for 30 s. Resuspend the beads in 50.5 mL Elution Buffer, heat seal, cool, and vortex. Incubate for 5 min at 20 C-25 C. p. Place on a magnetic plate stand and transfer 50 mL of sample to a new 96-well skirted PCR plate. q. Make the Ligation Master Mix. r. Add 50 mL Adaptor Ligation Mix to 50 mL of sample. s. Place the plate on the pre-warmed thermal cycler and run the following ''Ligation'' program: t. Add 80 mL SPRI beads to each sample well and incubate for 5 min at 20 C-25 C. u. Place on a magnetic plate stand and discard the clear supernatant. v. Add 200 mL 80% EtOH to each well avoiding the pellet, wait 30 s. w. Remove and discard EtOH. x. Repeat for a total of 2 washes. y. Centrifuge briefly and remove remaining EtOH. Air dry on a magnetic plate stand for 30 s. z. Resuspend the beads in 30.5 mL Elution Buffer, incubate for 5 min at 20 C-25 C. aa. Place on a magnetic plate stand and transfer 30 mL of sample to a new plate bb. Make Index PCR Master Mix. cc. Add 60 mL Index PCR Mix to each 30 mL sample. dd. Add 10 mL of an individual i7 sample index from the Single Index Kit T Set A to each well and record the well ID. ee. Run the ''Library Index Amplification'' PCR program on a thermal cycler: Pause point: store 2 C-8 C up to 72 h or À20 C for one week. Add 60 mL SPRI beads to each sample well, heat seal, cool, and vortex. Incubate for 5 min at 20 C-25 C. Place on a magnetic plate stand and discard 165 mL of the clear supernatant. jj. Add 180 mL 80% EtOH to the pellet. Wait 30 s kk. Remove and discard EtOH. ll. Repeat steps kk and ll for a total of 2 washes mm. Centrifuge briefly and remove remaining EtOH. Air dry on a magnetic plate stand for 30 s. nn. Resuspend the beads in 35.5 mL Elution Buffer, incubate for 5 min at 20 C-25 C. oo. Place on a magnetic plate stand and transfer 35 mL of sample to a new plate once the solution has cleared. pp. Make 1:10 dilution (45 mL Elution Buffer + 5 mL HTO Final Libraries) and record the barcode and well location. Timing: 3 h Sequencing. High quality libraries were generated . 18. Library QC and Sequencing a. Determine each library's average fragment size with a Fragment Analyzer. b. Determine each library's concentration using the Quant-iTä PicoGreenä dsDNA Assay or Kapa SYBR Fast qPCR Complete Assay for BioRadâ iCycler. See troubleshooting section problem 3 for potential solution to low library concentration. c. Based on the recommendations of the quantification assay used, calculate size-adjusted molarity (nM) based on the average base pair size obtained via QC for each library. d. Determine the targeted number of reads per each library, we recommend: i. 45K reads per cell for each Gene Expression Library. ii. 3K reads per cell for each HTO library. e. Using each well's size-adjusted molarity (nM) and targeted number of reads, pool a normalized concentration of each Gene Expression and HTO Library into a 1.5 mL low-retention, screw-cap tube. f. Dilute pool to desired concentration and volume for sequencing with Qiagen Buffer EB or similar Tris low EDTA buffer. i. Typically, 10 nM with a total volume of 150 mL. g. Determine the appropriate Illumina sequencing platform and flow cell size to sequence the pooled RNA and HTO library pools together with the following sequencing parameters: i. Paired-end, Single-Index ii. Read lengths: R1: 28 R2: 91 i7 Index: 8 iii. Include 1% PhiX control spike-in. 19. Storage and Freezing a. Using reagent guidelines provided on kit boxes and containers, place all reagents in the appropriate freezers. Store custom oligo stocks at 2 C-8 C for up to 1 month. Store the custom HTO i7 stock plate at À80 C for up to 3 months. Store the custom oligo working dilutions and working HTO i7 index plate at À20 C for up to 3 months. b. In the event the operator is no longer able to continue this protocol at any step where a safe stopping point and storage of libraries is not explicitly referenced, refer to 10x Genomics support for sample storage guidance. c. Store all the sequencing-ready libraries at À20 C for up to 1 year. High-dimensional flow cytometry sample preparation This section describes the flow cytometry procedure for staining and data acquisition of human peripheral blood mononuclear cells (PBMCs) with high dimensional immunophenotyping panels. This protocol has been optimized for longitudinal studies by incorporating features such as a bridging control sample in each batch for normalization, and commercial reagents for staining, washing, and fixation to reduce batch variation. Automated pipetting with the Integra VIAFLO 96 electronic pipette reduced processing time while maintaining high cell viability and retention. The Integra mixing operation programs were optimized to ensure sample pellets were fully resuspended to avoid variable staining due to clumping outlined in troubleshooting problem 4. The data displayed in this protocol is from a 25-color immune survey panel acquired on the Cytek Aurora five laser spectral cytometer but this method can be adopted for any flow cytometry experiment and instrumentation. CRITICAL: All incubations prior to sample fixation are at 2 C-8 C protected from light and sealed with a Microseal B adhesive seal. CRITICAL: All sample plate centrifuge steps are at 750g for 5 min at 2 C-8 C with swinging centrifuge buckets and aerosol containment covers. Note: The Integra VIAFLO 96 steps may be substituted with manual washing/aspiration using the same volumes noted in the protocol. This section details how to aliquot and stain samples to allow robust longitudinal flow cytometry analysis. a. Adjust the volume of each sample to 10 million viable per mL with DPBS. If the final volume is less than 120 mL, add DPBS so that there is at least 120 mL in the well. Note: Less than 120 mL can be used, as the samples will be centrifuged, and the supernatant aspirated before the first staining step. Ensure that each well receives the desired number of cells. b. Add 100 mL (equivalent to 1 million viable cells) of each sample to the corresponding labeled wells of the 96-well semi-skirted Eppendorf sample plate. Insert the sample plate into a precooled Integra PCR 96 well cooling block (2 C-8 C). c. Add 50 mL (equivalent to 0.5 million cells) of extra batch control cells to empty wells of the sample plate, or an additional plate, for staining single color controls. Make sure to include a negative control. Note: If single color controls have previously been recorded as Library Reference Controls in the SpectroFlo software, they do not need to be recorded again. The normalized reference controls will work for future batches of the same panel. Note: If single color controls are on a separate plate, repeat all steps for both the sample and control plates. Alternatives: If extra batch control samples are not available, other PBMC samples can be used for single color controls. If cells are not available, compensation beads can also be used, but must be optimized before running the experiment. d. Move the plate and cold block into the right most Integra position. Place a reservoir with 50 mL of cold DPBS into the middle Integra position. e. Run the Integra program ''FLOW_DILUTE'' to add 150 mL of DPBS to each sample well. f. Seal the plate with an adhesive seal and centrifuge at 750 g for 5 min at 2 C-8 C. a. Prepare the Viability and Fc Blocking solution in a 5 mL Eppendorf tube and store on ice protected from light for up to 15 min. b. Aliquot 100 mL of Viability and Fc Blocking solution to the wells corresponding to samples and Viability single color control in an Eppendorf 96-well full skirt plate. Store in the dark at 2 C-8 C. Note: Do not add the Viability and Fc Blocking Solution to all control wells, only the single color control for the Viability stain. c. Remove the sample plate from the centrifuge. Insert the plate into the cold block, and transfer to the right most Integra position. Remove the adhesive seal. d. Label a new reagent reservoir as biohazardous waste and place it in the middle position of the Integra deck. Run the Integra program ''FLOW_REMOVE'' to remove 250 mL of supernatant from sample wells. Note: Once the Integra aspiration step completes, immediately remove the waste reservoir from the middle integra position and discard the fluid in a waste bottle with 10% bleach solution. e. Place the 96-well full-skirt plate containing the Viability and Fc Blocking solution in the middle position on the Integra. f. Run the Integra program ''FLOW_RSP_MM'' to add 90 mL of Viability and Fc Blocking solution to each sample and mix ten times. g. Incubate for 30 min at 2 C-8 C protected from light. h. Label a new reagent reservoir as Cell Staining Buffer and place it in the middle position of the Integra deck. Add 50 mL of Cell Staining Buffer to the reservoir. Insert the sample plate on the cold block, remove the seal and place on the right of the stage on the Integra. i. Run the Integra program ''FLOW_DILUTE'' to add 150 mL of Cell Staining Buffer to each sample well. j. Seal the plate and centrifuge at 750 g for 5 min at 2 C-8 C. CRITICAL: Centrifuge antibody storage vials at 10,000g for 10 min prior to adding to mastermix to reduce antibody aggregates outlined in troubleshooting problem 5 . a. Record the lot number of each antibody vial and note lot changes between batches. b. Keep antibodies protected from light at 2 C-8 C until needed. c. Prepare the antibody master mix solution in a 5 mL Eppendorf tube (Table 1) and place on ice, protected from light for up to 1 h. d. Vortex the antibody master mix tubes for 10 s. Centrifuge the tubes at 3000 g, 2 C-8 C for 2 min. e. Transfer 90 mL master mix into corresponding wells of a 96-well full-skirt plate. Keep the master mix solution plate at 2 C-8 C protected from light for up to 1 h until staining. ll f. Insert the sample plate on the cold block and remove the adhesive seal. Place the biohazardous waste reservoir in the middle position of the Integra deck. g. Run the Integra program ''FLOW_REMOVE'' to remove 250 mL of supernatant from sample wells. Note: Once the Integra aspiration step completes, immediately remove the waste reservoir from the middle integra position and discard the fluid in a waste bottle with 10% bleach solution. h. Place the 96-well full-skirt plate containing 100 mL of antibody master mix in each well needed in the middle position on the Integra. i. Run the Integra program ''FLOW_RSP_MM'' to add 90 mL of antibody master mix to the sample wells and mix ten times. j. Add the corresponding antibody volume to each single color control well. Note: The volume of each antibody added to single color controls should be the same as the volume for a single sample, including the 20% overage. k. Seal the plate and incubate for 30 min at 2 C-8 C protected from light. l. Insert the sample plate on the cold block and remove the adhesive seal. Place the reagent reservoir containing Cell Staining Buffer in the middle position of the Integra deck. m. Run the Integra program ''FLOW_DILUTE'' to add 150 mL of Cell Staining Buffer to each sample well. n. Seal the plate and centrifuge at 750 g for 5 min at 2 C-8 C. o. Insert the plate into the cold block and remove the adhesive seal. Place the waste reservoir in the middle position of the Integra deck. p. Run the Integra program ''FLOW_REMOVE'' to remove 250 mL of supernatant from sample wells. Remove the waste reservoir from the middle integra position and discard the supernatant in a waste bottle with 10% bleach solution. q. Place the reagent reservoir containing 50 mL Cell Staining Buffer in the middle position of the Integra deck. r. Run the Integra Program ''FLOW_WASH'' to add 250 mL of Cell Staining Buffer to each sample well and mix ten times. s. Seal the plate and centrifuge at 750 g for 5 min at 2 C-8 C. t. Place the sample plate on the cold block, remove the seal and place it on the right of the stage on the Integra. Place the waste reservoir in the middle position of the Integra deck. u. Run the Integra program ''FLOW_REMOVE'' to remove 250 mL of supernatant from sample wells. Note: Once the Integra aspiration step completes, immediately remove the waste reservoir from the middle integra position and discard the fluid in a waste bottle with 10% bleach solution. v. Place the reagent reservoir containing 50 mL Cell Staining Buffer in the middle position of the Integra deck. w. Run the Integra Program ''FLOW_WASH'' to add 250 mL of Cell Staining Buffer to each sample well and mix ten times. x. Seal the plate and centrifuge at 750 g for 5 min at 2 C-8 C. Note: Two full washes are needed after antibody master mix staining to ensure no excess antibody is in the solution when the cells are fixed as this could lead to non-specific attachment of antibody to the cell surface. y. Insert the plate on the cold block and remove the adhesive seal. Place the waste reservoir in the middle position of the Integra deck. z. Run the Integra program ''FLOW_REMOVE'' to remove 250 mL of the supernatant from sample wells. Note: Once the Integra aspiration step completes, immediately remove the waste reservoir from the middle integra position and discard the fluid in a waste bottle with 10% bleach solution. 23. Fix Stained Cells a. Remove the waste reservoir from Integra stage and place a 96-well full-skirt plate containing 110 mL of 20 C-25 C FluoroFix Buffer in each well in the middle position on the Integra. CRITICAL: FluoroFix -BioLegend: 4% p-formaldehyde -acute toxicity. Note: Other 4% p-formaldehyde fixation buffers may be used in place of FluoroFix Buffer. b. Run the Integra program ''FLOW_FIX'' transfer 100 mL of FluoroFix Buffer from the fixation plate to the sample plate and mix ten times. c. Incubate for 30 min protected from light at 20 C-25 C. d. Insert the sample plate on the cold block and remove the adhesive seal. Place the reagent reservoir containing Cell Staining Buffer in the middle position of the Integra deck. e. Run the Integra program ''FLOW_DILUTE'' to add 150 mL of Cell Staining Buffer to each sample well. f. Seal the plate with an adhesive seal and centrifuge at 750 g for 5 min at 2 C-8 C. g. Place the plate on the cold block and remove the adhesive seal. Place the waste reservoir in the middle position of the Integra deck. h. Run the Integra program ''FLOW_REMOVE'' to remove 250 mL of supernatant from sample wells. Note: Once the Integra aspiration step completes, immediately remove the waste reservoir from the middle integra position and discard the fluid in a waste bottle with 10% bleach solution. i. Place the reagent reservoir containing 50 mL Cell Staining Buffer in the middle position of the Integra deck. j. Perform a post-fixation wash by running the Integra Program ''FLOW_WASH'' to add 250 mL of Cell Staining Buffer to each sample well and mix ten times. k. Seal the plate with a Microseal B adhesive seal and centrifuge at 750 g for 5 min at 2 C-8 C. Note: Only one wash is needed because the fixative has been diluted beyond efficacy. l. Place the sample plate on the cold block, remove the seal and place on the right of the stage on the Integra. Place the waste reservoir in the middle position of the Integra deck. m. Run the Integra program ''FLOW_REMOVE'' to remove 250 mL of supernatant from sample wells. Note: Once the Integra aspiration step completes, immediately remove the waste reservoir from the middle integra position and discard the fluid in a waste bottle with 10% bleach solution. n. Place the reagent reservoir containing 50 mL Cell Staining Buffer in the middle position of the Integra deck. Run the Integra program ''FLOW_FINAL'' to add 100 mL of Cell Staining Buffer and mix ten times to resuspend the cells. o. Seal the plate with an adhesive seal and cover with aluminum foil and store at 2 C-8 C. Pause point: Cells are fixed at this stage and may be stored at 2 C-8 C for up to 18-24 h before data acquisition. Timing: 4 h (for a full 96 well plate) This section includes information on settings used to acquire processed samples on a five laser Cytek Aurora. CRITICAL: Acquire data within 24 h of staining and fixing cells. a. Remove the seal from the sample staining plate. Mix the wells 10 times to resuspend the samples. b. Transfer the 100 mL of sample volume from each well to a 96 well U bottom plate. c. Add 160 mL of Cell Stain Buffer to the original sample staining plate and mix 10 times. Transfer 160 mL of the wash volume to the 96 well U bottom plate for acquisition. Note: The total volume in the 96 well U bottom plate should be 260 mL per well. 25. Set up Cytek Aurora cytometer a. Startup and QC instrument according to manufacturer's recommendations in plate mode. Note: Using the same QC bead lot throughout the course of a longitudinal study will improve consistency. It is recommended that you purchase enough of the same bead lot for the duration of your study. b. Set the instrument to acquire 200 mL of sample from each well, mix each well before acquisition, and backflush the sample probe 2 times after every well. Note: These settings will allow for $50 mL of dead volume per well and acquire samples at a rate of 5000 events/sec or less. This should prevent any bubbles from entering the stream and causing artifacts in the data. Use of the outlined cell thawing and counting protocol showed consistent cell recovery and viability values for the batch control leukopak PBMC sample over a period of 1 year composed of 42 batches ( Figure 10 ). The expected output of this protocol is high quality longitudinal flow cytometry data over multiple batches. With a starting input of one million cells, the typical recorded number of cells from the cytometer is $400,000 cells. Recording a high number of cells improves the counts and statistical significance of rare cell types (Figures 14 and 15 ). This protocol provides a method for generating high quality longitudinal flow cytometry data from fixed human PBMCs. This method may be adapted for other tissue types, but development is needed to ensure the same quality of data over time. This protocol has been optimized for scRNAseq data generation from human PBMC with cell viability greater than 70%. The number of samples for each pool is limited to the number of unique HTOs and HTO i7 indexes available. The number of samples per each pool is also limited by sequencing capacity on a NovaSeq S4 flowcell which is only able to sequence 12 GEX libraries and 12 corresponding HTO libraries at the appropriate depth. Problem 1 Cellaca MX cell counter. Cells appear out of focus, or the Green Fluorescence signal FS1 is dull when viewing loaded cell counter plate (cell count and normalization step 6g), shown in Figure 16 . Stop the preview and select Auto-Focus. Preview BR1 and FL1 again to confirm the cells are in focus. If necessary, adjust the focus further using the fine and coarse focus adjustments. Note: The focus can only be adjusted when the preview has been stopped. A new preview must be done each time the focus is adjusted. Problem 2 scRNA-seq low cell viability. If a sample's cell viability is too low (usually below 50%) when reviewing cell viability results (cell hashing, gem generation, and reverse transcription step 11b) it is recommended not including that sample in the pool for GEM generation. Adjust the pooling calculation for the batch control accordingly. Dropping a sample with low viability ensures the correct number of cells are loaded per sample and maintaining high quality single-cell data. If a second aliquot of this sample is available, thaw, count and replace. If this sample also has low viability, consider sequencing this sample separately through standard 10X 3 0 scRNA seq without cell hashing. Problem 3 scRNA-seq low HTO product, high TSO product. It can be difficult to separate the amplified HTO libraries from excess TSO if the product concentration is low when reviewing library concentration (library QC and sequencing step 18b). If an HTO library shows low amplified product and high TSO product, an additional amplification can be run with illumina P7 and P5 adapter primers at a final concentration of 0.1 mM with 3-5 cycles of amplification. High dimensional flow cytometry, poor quality fluorescence data. Suboptimal resuspension of cell pellets in the antibody mastermix may lead to streaking of fluorescent signals observed when reviewing acquired flow cytometry data (high dimensional flow cytometry step 27f), shown in Figure 17 . After centrifuging the PBMC samples, cell pellets will not break up completely without proper mixing. If pellets are not well dispersed, some cells are not fully stained, resulting in a signal that streaks towards zero on the axis. This mixing effect is related to how well each pellet is resuspended in the well, so it may appear to occur in random samples. Increasing the number of mixes in each step, particularly during the antibody staining step and using standard width pipette tips instead of Wide Bore tips, will help prevent this mixing effect from impacting data quality. The Integra mixing operation programs were optimized (Flow Cytometry Cell Staining steps 1-4) to ensure sample pellets were fully resuspended to avoid variable staining due to clumping. High dimensional flow cytometry presence of antibody aggregates. High signal artifacts appear above fully stained populations, sometimes appearing with new antibody lots, when reviewing acquired flow cytometry data (high-dimensional flow cytometry step 27f), shown in Figure 18 , resolved in Figure 19 . This issue is most likely to happen with BD Brilliant Ultra Violet (BUV) fluorophores. High signal artifacts are typically from antibody aggregates in the storage vial. Spinning down the vials at 10,000 g for 10 min at 2 C-8 C before aliquoting from them can help prevent aggregates Figure 18 . BUV805 CD11c staining across 6 batches over time After the first 3 batches, a new antibody lot was used that introduced high signal artifacts to the data. from staining the sample. The vials were originally centrifuged in a small table top centrifuge at 2,000 g for 30 s, but this was insufficient to remove aggregates whereas 10,000 g for 10 min removed most of the aggregates as reflected in samples stained with various Brilliant Ultra Violet (BUV) dyes (Figure 19 ). To ensure that antibody aggregates are minimized, this centrifugation step was included in Flow Cytometry Cell Staining step 22. Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Julian Reading (julian.reading@alleninstitute.org). This study did not generate new unique reagents. The published article includes all data sets/code generated or analyzed during this study. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Multimodal analysis for human ex vivo studies shows extensive molecular changes from delays in blood processing Cell Hashing with barcoded antibodies enables multiplexing and doublet detection for single cell genomics Simultaneous trimodal single-cell measurement of transcripts, epitopes, and chromatin accessibility using TEA-seq Longitudinal immune dynamics of mild COVID-19 define signatures of recovery and persistence The protocol Graphical Abstract was created using BioRender.com. The authors thank Leila Shiraiwa and Ernie Coffey for operational support. We are grateful to the Allen Institute for Immunology for funding the development of this protocol. The authors also wish to thank the Allen Institute founder, Paul G. Allen, for his vision, encouragement, and support. The authors declare no competing interests.