• Before starting
• Documentation
• What you’ll need
• 10X reagents (included):
• Not included in the 10X kits:
• Day 1
• Cell Preparation
• Cell Labeling
• Cell suspension volume calculator table
• Encapsulation
• Transfer and GEM-RT Incubation
• Day 2
• Post GEM-RT Cleanup
• cDNA Amplification, Cleanup, and QC

Before starting

Documentation

We use the 10X Chromium Controller platform to encapsulate and barcode single cells. The following protocol describes the steps for single cell mRNA-seq combined with CITE-seq and cell hashing, but the platform is capable of many other preparations. Refer to the corresponding protocols for these and ensure you are using the appropriate protocol and reagents for your experiment. This protocol is based on the BioLegend TotalSeq Protocol using A Antibodies and the 10X Single Cell 3' v3.1 User Guide. The New York Genome Center's CITE-seq and cell hashing protocol is another useful resource. This protocol is not compatible with TotalSeq's B or C antibodies or 10x's Feature Barcoding Kit.

CITE-seq is a technique that allows simultaneous characterization of protein and RNA expression by labeling cells with antibodies conjugated to oligos. Cell hashing uses oligo conjugated antibodies to multiplex samples based on cell surface features. After cell preparation and labeling, this protocol follows the typical single mRNAseq workflow until the cDNA amplification step, where ADT (Antibody Derived Tag) and/or HTO (Hash Tag Oligonucleotide) primers are added to increase yield of products for CITE-seq and cell hashing, respectively. The amplification products undergo a bead cleanup, where the library preparations split. The supernatant fraction contains what will become the ADT and HTO libraries and the pellet contains what will become the mRNA libraries.

What you’ll need

10X reagents (included):

• Chromium Next GEM Single Cell 3ʹ GEM, Library & Gel Bead Kit v3.1 (2 boxes in -20C Freezer and 1 box in -80C Freezer)
• Chromium Next GEM Chip G Single Cell Kit (Room Temp)
• Single Index Kit T Set A (-20C Freezer)

Not included in the 10X kits:

• Cell Suspension (See following Instructions)
• 50% glycerol solution (Room Temp)
• Nuclease-free water (Room Temp)
• 10% Tween 20 (Room Temp)
• Buffer EB (Room Temp)
• SPRI Select Reagent (Room Temp)
• KAPA HiFi HotStart Ready Mix (-20C Freezer)
• ADT additive primer, 0.2uM stock (for CITE-seq)
• HTO additive primer, 0.2uM stock (for Cell Hashing)
• TruSeq Small RNA RPIx index primers, 10uM stock (for CITE-Seq)
• TruSeq D70x_ Long index primers, 10uM stock (for Cell Hashing)

Day 1

Cell Preparation

The optimal input cell concentration is 700-1,200 cells/µl. Depending on your concentration, you will need 2.1-23.6 uL of cell suspension as input for each sample. Refer to the Cell Suspension Volume Calculator Table at the end of this section.

While a high concentration of cells is desirable, it also increases the rate of ‘multiplets’. Multiplets occur when a single oil droplet contains 1 barcoded bead and two or more cells. Since each bead has a single barcode, transcripts from multiplets will appear as though they are from the same cell. 10X Genomics recommends 700-1,200 cells/µl in order to maximize cell encapsulation while avoiding excessive multiplets.

For more detail on cell preparation, refer to 10X’s literature, which covers:

• General preparation for limited samples
• Preparation from cultured cell lines
• General workflow
• Other details

Cell Labeling

The BioLegend TotalSeq protocol details cell labeling. You will need a CITE-seq antibody, usually a Totalseq A antibody from BioLegend. If you are doing cell hashing, you will also need a Cell Hashing antibody.

Cell suspension volume calculator table

Encapsulation

• Allow the Single Cell 3’ v3.1 Gel Beads to come to room temperature for 30 minutes before loading the chip.
• Thaw RT Reagent B and Reducing Agent B at room temp.
• If using a new kit: resuspend Template Switch Oligo in 80uL Low TE Buffer and leave at room temp for at least 30 minutes. If NOT using a new kit, retrieve Template Switch Oligo (already resuspended in TE Buffer) from -80C storage and allow to equilibrate to room temp for at least 30 minutes.
• Place RT Enzyme C on ice.
• Prepare Master Mix on ice, per sample (10% extra has already been included):

• Add 31.8 uL Master Mix into each tube of a PCR 8-tube strip. Keep these tubes on ice.
• Assemble the Next GEM Chip G by inserting it in the Secondary Holder under the guide (on the left side of the holder when open). The chip label should be at the top and facing toward you. Gently press down the right side of the chip until it clicks into place. Close the lid of the holder over the chip.

• Refer to the Cell Suspension Volume Calculator Table. Add the appropriate volume of nuclease free water to the Master Mix, then add the corresponding volume of cell suspension to the Master Mix, for a total of 75 µl in each tube. Gently pipette to mix the cells suspension before adding to the Master Mix. DO NOT ADD WATER DIRECTLY INTO THE CELL SUSPENSION.
• Load Row Labeled 1 with 70uL Master Mix without introducing bubbles.
• To prepare the gel beads, place the gel bead strip into the 10x Vortex Adapter. Vortex the gel beads in the adapter for 30 seconds, then remove the gel bead strip from the holder and centrifuge ~5 seconds. Place the gel bead strip back in the holder and attach the lid.
• To load Row Labeled 2, puncture the foil seal of the gel bead tubes and aspirate and dispense 50uL gel beads without introducing bubbles. Wait 30 seconds.
• Load Row Labeled 3 with 45uL partitioning oil, avoiding bubbles. Your chip should now look like the image below, with the unused wells filled with appropriate volumes of 50% glycerol if loading less than 8 samples.

• To attach the 10x gasket, align the notch with the top left-hand corner. Ensure the gasket holes are aligned with the wells. The smooth side of the gasket should be facing toward the wells. Do not touch the smooth side or press down on the gasket. Keep the assembly horizontal.
• Bring the assembly over to the 10x Chromium Controller. Turn on the machine and press the eject button when it appears on the screen.
• Place the assembled chip with the gasket in the tray, ensuring that the chip stays horizontal. The chip should be placed face up, with the notched corner in the upper left and the chip label at the top. Press the button to retract the tray. Confirm the Chromium Chip G program on screen. Press the play button. At completion of the run (~18 min), the Controller will chime. Immediately proceed to the next step.

Transfer and GEM-RT Incubation

• Place a tube strip on ice
• Press the eject button of the Controller and remove the chip. Turn off the Controller after use.
• Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees.
• Check the volume in rows labeled 1-2. Abnormally high volume in any well indicates a clog.
• Slowly aspirate 100 μl GEMs from the lowest point of each recovery well in the top row labeled 3 without creating a seal between the tip and the bottom of the well.
• As you aspirate the GEMs, check their appearance in the pipette tip. GEMs should appear mostly opaque and consistent white. Excess Partitioning Oil (clear) in the pipette tip indicates a potential clog.
• Over the course of ~20 sec, dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes.
• Attach the deep well head to the thermocycler, if not already in place. Incubate samples in the thermocycler under the protocol 10x scRNAseq->GEM INCUBATE (53C for 45min, 85C for 5min, 4C hold, lid 53C, 125uL volume).
• If doing the entire protocol in 3 days, end Day 1 here. Store at 4°C for up to 72 h or at −20°C for up to a week.

Day 2

Post GEM-RT Cleanup

• Equilibrate Reducing Agent B, cDNA primers, Dynabeads and Tapestation HSD5000 screentape and reagents to room temp for at least 30 minutes before use.
• Place Amp Mix on ice.
• Thaw Cleanup Buffer on heatblock at 65C for 10min, vortexing at 5min and 10min. Check that there are no visible crystals and vortex more if needed.
• Add 125μl Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex the biphasic mixture. Wait 2 min. This should separate into a clear aqueous phase on top and a pink layer on bottom with no opaque emulsion. A smaller aqueous phase volume indicates a clog during GEM generation.
• Slowly remove and discard 125 μl Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample. There will still be a small remaining volume of pink Recovery Agent/Partitioning Oil after removal. Observe these volumes across your samples as abnormal volumes may indicate a wetting failure or clog during encapsulation. Below is an example of successful samples.

• Prepare Dynabeads Cleanup Mix per sample (10% extra has already been included):

• Vortex Dynabeads Cleanup Mix and add 200uL to each sample. Pipette to mix 10 times.
• Incubate for 10 min at room temp. Pipette to mix again at 5 min after start of incubation. You should end up with a large brown layer of Dynabeads Cleanup Mix at the top and a smaller white Recovery Agent/Partitioning Oil layer below. Observe these volumes across your samples as abnormal volumes may indicate a wetting failure or clog during encapsulation. Below is an example of successful samples.

• Prepare Elution Solution I per sample (10% extra has already been included):

• Vortex and centrifuge Elution Solution briefly.
• At the end of the 10min incubation, place the sample tube strip on a 10x Magnetic Separator in the HIGH position until solution clears (~5 min).
• Remove and discard the supernatant.
• Keeping the samples on the magnet, add 300uL 80% ethanol.
• Wait 30 sec, then remove the ethanol.
• Keeping the samples on the magnet, add 200uL 80% ethanol.
• Wait 30 sec, then remove the ethanol.
• Centrifuge briefly. Place on the magnet on LOW
• Remove remaining ethanol. Air dry for 1 min.
• Remove from the magnet. Immediately add 35.5 μl Elution Solution I.
• Pipette to mix (pipette set to 30 μl) without introducing bubbles.
• Incubate 2 min at room temperature.
• Place on the magnet on LOW until the solution clears.
• Transfer 35 μl sample to a new tube strip.

cDNA Amplification, Cleanup, and QC