Before starting

Documentation

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

Sequencing Guidelines

Normalize and Pool

1. 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.
2. 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.
3. 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.
4. 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.
5. 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.