We use the 10X Chromium X Controller platform updated with firmware v2.0 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 the TotalSeq B Mouse v1.0 Antibody cocktail Protocol and the 10X Single Cell 3' v4 User Guide. The New York Genome Center's CITE-seq and cell hashing protocol is another useful resource.
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 Feature Barcode primers are added to increase yield of products for CITE-seq. The amplification products undergo a bead cleanup, where the library preparations split. The supernatant fraction contains what will become the Cell Surface Protein libraries and the pellet contains what will become the mRNA libraries.
Protocol
‣
Part 1: Staining and Encapsulating Cells
Staining and Encapsulating Cells
‣
Reagents For Cell Staining
Reagent
Manufacturer
Cat No.
Location
TotalSeq B Antibody Cocktail
Biolegend
199902
4°C Fridge
Cell Staining Buffer
Biolegend
420201
4°C Fridge
TruStain FcX PLUS (anti-mouse CD16/32)
Biolegend
156603
4°C Fridge
‣
Reagents For 10X Cell Preparation
Reagent
Manufacturer
Cat. No.
Storage Location
Thaw Condititons
GEM-X Single Cell 3’ Gel Bead v4
10X Genomics
2001128
-80°C Freezer
Equilibrate to room temperature 30 min before loading the chip
RT Reagent E
10X Genomics
2001106
-20°C Freezer
Vortex, verify no precipitate, centrifuge briefly. If a precipitate is observed, warm the tube with hands until the precipitate dissolves. Vortex, centrifuge briefly.
Template Switch Oligo B
10X Genomics
2001027
-20°C Freezer or -80°C Freezer if resuspended
If starting with a new vial: Centrifuge briefly and resuspend in 65 µL of Low TE Buffer. Vortex 15 sec and centrifuge briefly. After resuspension store at -80°C.
If already resuspended: thaw at room temperature. Vortex and centrifuge briefly.
Reducing Agent B
10X Genomics
2000087
-20°C Freezer
Vortex, verify no precipitate, centrifuge briefly
RT Enzyme E
10X Genomics
2001105/2001146
-20°C Freezer
Store until need. Centrifuge briefly. Keep on ice while handling. Return immediately to the freezer after use.
1x PBS
-
-
Ambient
Aliquot 100 µL in an Eppendorf LoBind tube and place on ice.
‣
Other Reagents and Materials
Reagent/Material
ManufacturerC
Cat. No.
Storage Location
Notes
Partitioning Oil B
10X Genomics
2001213
Ambient
Kit Specific. Do not use partitioning oil from non-GEM-X kits
GEM-X 3’ Chip
10X Genomics
2001097
Ambient
X/iX Chip Gasket
10X Genomics
3000656
Ambient
Bue gasket
Chromium X/iX Chip Holder
10X Genomics
3000598
Ambient
Black Chip Holder
10X Vortex Adapter
10X Genomics
330002
Ambient
-
40 µm Flowmi Cell strainer
Flowmi
Ambient
-
50% glycerol
Ricca
3290-32
Ambient
If performing less than 8 reactions
LoBind DNA Tubes
Eppendorf
022431021
Ambient
Use for all 10X master mixes
USA Scientific Temp-Assure PCR Strip Tubes
USA Scientific
1402-4700
Ambient
Use for all 10X samples and PCR reactions
Rainin Universal Tips
METTLER-TOLEDO RAININ LLC
-
Ambient
Use for all pipetting through the conclusion of the encapsulation including during cell staining.
‣
1. Lyophilized Panel Reconstitution
Equilibriate the lyophilized panel vial(s) to room temperature for 5 minutes. One panel is needed per sample.
Place lyophilized panel vial in an empty 1.5mL tube, spin down at 10,000 x g for 30 seconds at room temperature.
Rehydrate lyophilized panel by adding 27.5 µL of Cell Staining buffer. Replace the cap and vortex for 10 seconds.
Incubate at room temperature for 5 minutes.
Vortex again for 10 seconds and spin down for 30 seconds at 10,000 x g at room temperature.
Transfer the entire volume (27.5 µL) of reconstituted cocktail to an Eppendorf LoBind Tube.
Centrifuge at 14,000 x g for 10 minutes at 4°C.
While cocktail is centrifuging, resuspend and block cells. Once cocktail is finished centrifuging, immediately place on ice until ready to add to the blocked cells.
‣
2. Block and Stain Cells
Transfer 500 µL of the single cell suspension with a concentration of 1,000,000 cells/mL to a 12x75 mm culture tube. Spin cells for 5 minutes at 4°C at 300 x g to pellet cells. Remove the supernatant without disturbing the cell pellet.
For mouse cells: resuspend cell pellet in 24.75 µL of cell staining buffer and add 0.25 µL TruStain FcX PLUS blocking reagent. Gently pipette mix. Incubate on ice or at 4°C for 10 minutes.
Transfer 25 µL of the reconstituted cocktail from step 1.8 to the 25 µL of FcR blocked cells. The final staining volume is 50 µL.
Incubate cells on ice or at 4°C for 30 minutes. At the end of this incubation, retrieve all 10X reagents to that need to equilibrate to room temperature. Keep RT Enzyme stored in the freezer until just before use.
‣
3. Wash, Filter, and Count Cells
At the end of the 30 minute incubation, add 3 mL of Cell Staining Buffer to the cells and spin at 4°C for 5 minutes at 300 x g.
Repeat this wash 2 more times for a total of 3 washes.
Resuspend cells in up to 500 µL of cell staining buffer. If cell numbers are a concern, resuspend in a lower volume of cell staining buffer.
Slowly filter the entire volume through a 40 µm Flowmi Cell Strainer into a new LoBind Eppendorf Tube.
Verify cell concentration and viability after filitration. The optimal number of cells is 700 - 1,200 cells/µL for targeting 10,000 cells and 1,300 - 1,600 cells/µL for targeting 20,000 cells.
Only 10 µL of the product from step 2.3a of the protocol is necessary for Gene Expression Library preparation. Calculate the total yield by multiplying the concentration by 40 µL. Divide the total yield by 4 in order to determine the amount used and to determine number of sample index PCR cycles. The remaining cDNA can be stored at -20°C for subsequent use.
Only 5 µL of the product from step 2.3b of the protocol is necessary for Cell Surface Protein library preparation. The remaining cDNA can be stored at -20°C for subsequent use.
For Cell Surface library construction, the optimal number of Sample Index PCR cycles may vary. We have found when targeting a larger number of cells, only 8 cycles is necessary for amplifying the libraries.
Sequencing Guidelines
Normalize and Pool
Quantify each of your libraries on Qubit. For most libraries, using the HS dsDNA Qubit assay with 2uL of input will yield a reading. Record the concentration in ng/uL for each library. Libraries targeting a larger number of cells, or with high RNA content make produce very concentrated libraries. In these case, libraries can be dilluted 1:10 in Buffer EB before measuring on Qubit or running on the Tapestation.
If not already completed, run your libraries on Tapestation with the High Sensitivity D5000 assay. Remember to allow Tapestation reagents to sit at room temperature for at least 30 minutes before use. Save your Tapestation results by going to File -> Create Report -> Save as pdf. This file can then be emailed or uploaded to Asana. For the base pair length, we usually use the value of the peak identified by the Tapestation analysis software. This value is shown both on the tracing itself and in the Peak Table for each sample.
For sequencing, 10X recommends a sequencing depth of 20,000 read pairs per cell and 5,000 read pairs per cell for Gene Expression Libraries and Cell Surface Protein libraries respectively. Libraries of different types can pooled together. 10X recommends pooling with a ratio of 4:1 for Gene Expression Libraries : Cell Surface Libraries.
Download our 10X nM Conversion Calculator here. Enter the concentration (from Qubit) and the base pair length (from Tapestation) in the appropriate cells and it will give you the nM concentration for each library. You also need the Target Cell Recovery values. Normalize and pool all your libraries to 2 nM in a LoBind Eppendorf tube. If you need to dilute your libraries, we recommend using at least 2uL to minimize pipetting errors. The example sheet of the calculator provides further detail.
Quantify your pool on Qubit and run on the Tapestion. Enter into the calculator sheet to check that your pool is close to the 2 nM target concentration.
Loading the Sequencer
The next step is to dilute and denature the prepared libraries. Illumina’s general guidelines for this on the NextSeq can be found here.
Illumina’s system guide for the NextSeq, which covers the sequencing workflow, can be found here.
