HTG 10X Genomics 3’ Gene Expression

1. How do I schedule an appointment for a 10X Genomics experiment?

Appointments for 10X Genomics experiments can be scheduled with Opal Allen (opal.allen@hci.utah.edu; 801-581-6346) at the High-Throughput Genomics Shared Resource. Three time slots (9 am, 11 am, and 1 pm) are available on Monday through Thursday of each week. We recommend finalizing the appointment at least two to three weeks in advance to ensure that the desired date and time slot are available for your experiment.

2. Can I cancel a 10X Genomics appointment due to poor cell viability, low cell concentration, or a failure of cells to achieve the desired growth state?

Researchers can cancel a 10X Genomics appointment. Please contact Opal Allen at the HTG Shared Resource as soon as you are aware of the necessity to cancel.

3. Can I perform a practice cell preparation and acquire feedback from the HTG Shared Resource in regards to the quality of the cell suspension and viability of the cells?

We strongly encourage researchers to perform a practice cell preparation prior to their first single cell experiment on the 10X Genomics platform.

4. What learning resources are available for sample preparation on the 10X Genomics website?

Technical notes and sample preparation protocols for 3’ gene expression experiments can be reviewed and downloaded from the 10X Genomics website.

Instructional videos that describe the library preparation process are also available.

5. What recommendations are available for preparing a cell suspension?

The 10X Genomics Cell Preparation Guide describes best practices and methods for washing, concentrating, resuspending, and counting cells.

6. Which buffer should be used to resuspend cells in preparation for a 10X Genomics Single Cell RNA sequencing experiment?

Cells should be washed a minimum of two times and resuspended in 1X PBS with 0.04% BSA. Cells should always be maintained at a concentration below 5,000 cells/µL to reduce the incidence of cell aggregation. Resuspension of cells should be performed by slow pipetting using wide bore pipette tips.

7. How many samples can be loaded in a 10X Genomics Chromium Chip B?

Chromium microfluidic chips are one-time use consumables that can simultaneously partition up to eight samples per run.

8. What cell diameter is compatible with the 10X Genomics Single Cell platform?

The microfluidic channel within a 10X Genomics Chromium Chip B is approximately 50-60 µm in diameter which provides a theoretical upper limit of the cell size that is compatible with the platform.

9. What is the optimal cell concentration for a 10X Genomics RNA sequencing experiment?

The optimal cell concentration for a 10X Genomics single cell RNA sequencing experiment is 400-1200 cells/µL in a minimal volume of 30-100 µL. A 5 µL aliquot of the cell suspension will be used to measure the cell concentration using a Countess II FL Automated Cell Counter.

10. How should I measure the concentration of cells?

Cell concentration can be measured using a Fisher Scientific Countess II FL Automated Cell Counter (cat# AMQAX1000). A 5 µL volume from a cell suspension is combined with Trypan Blue to stain cells and provide information regarding the cell concentration, cell viability, and cell diameter.

11. How many cells are needed for a 10X Genomics Single Cell RNA sequencing experiment?

Single cell RNA sequencing experiments routinely target the retention of 500 to 10,000 cells which require an input of 800 to 16,000 cells. Approximately 40-65% of cells loaded into the 10X Genomics Chip B device will be partitioned and recovered. However, when a high percent of dead cells is present within a cell suspension, an apparent lower cell recovery rate will likely result in the sequencing data.

12. How is single cell resolution achieved with the 10X Genomics single cell platform?

A 34 µL volume of suspended cells is dispensed into a 10X Genomics Chip B at a dilution such that the majority (90-99%) of emulsion droplets that are formed following passage through the microfluidic device are absent of cells. The remaining droplets are largely occupied by a single cell. A low rate of multiplet occupancy will be experienced in single cell experiments and this rate is observed to increase when higher quantities of cells are loaded into the Chip B partitioning device.

13. What is the estimated doublet rate in a 10X Genomics single cell experiment?

The percentage of emulsion droplets containing two or more cells is influenced by the quantity of cells loaded into the Chip B partitioning device and the prevalence of cell aggregates within the cell suspension. Assuming the absence of cell aggregates, the following table, adopted from the 10X Genomics User Guide, can be used to estimate the occurrence of multiplet partitions within an emulsion.

Multiplet rate (%)	# of Cell Loaded	# of Cell Recovered
0.40%	800	500
0.80%	1,600	1,000
1.60%	3,200	2,000
2.30%	4,800	3,000
3.10%	6,400	4,000
3.90%	8,000	5,000
4.60%	9,600	6,000
5.40%	11,200	7,000
6.10%	12,800	8,000
6.90%	14,400	9,000
7.60%	16,000	10,000

14. How should I remove aggregates from my cell suspension?

A 40 µm Flowmi Cell Strainer (Bel-Art cat#H136800040) can be used to reduce the prevalence of aggregates within a cell suspension. The Flowmi device can be used with low cell volumes, however, the cell concentration may be reduced by approximately 20-40% following passage through the strainer.

15. What methods are available to remove dead cells?

Both live and dead cells must be considered when estimating the anticipated doublet rate that will be obtained following partitioning of a cell suspension for a 10X Genomics experiment. Therefore, a high percentage of dead cells will impact the targeted number of live cells that can be acquired for single cell analysis. 10X Genomics provides a demonstrated protocol for dead cell removal.

16. What does the term “wetting failure” mean?

A wetting failure is characterized by the absence of a uniform emulsion in an outlet well of a microfluidic chip following cell partitioning. A wetting failure leads to a loss of proper partitioning of single cells and reagents. Common causes of wetting failures include incorrect priming of reagents in the microfluidic channels, presence of air bubbles in the bottom of wells in the microfluidic chips, presence of surfactant or other viscous reagents in the cell media, or excessive presence of cell aggregates. Approximately 1% of experiments may experience a wetting failure in spite of proper loading of the microfluidic chip.

17. How frequently are wetting failures experienced at the HTG Shared Resource?

The HTG Shared Resource experiences wetting failures in approximately 1.25% of experiments. That being said, approximately 80% of the wetting failures have been associated with a single user working with a single cell type. We hope researchers will carefully consider cell preparation methods that reduce cell debris and aggregates such as to minimize the occurrence of wetting failures.

18. What costs are associated with a wetting failure?

10X Genomics will cover the reagent costs associated with any microfluidic channel that experiences a wetting failure. Provided that excess sample is available, cell partitioning will be repeated for any sample that fails to form a proper emulsion on the first pass. However, if additional sample is not available, adjacent wells containing other samples that formed a proper emulsion will be processed through library prep as we are unable to replace the reagents used for those samples that successfully formed an emulsion.

19. Can I recover my sample from the microfluidic Chip B in the event of a wetting failure?

Unfortunately, cell suspensions cannot be recovered from microfluidic chips following the event of a wetting failure.

20. How is the 10X Genomics Reverse Transcription primer used to enable the identification of molecules within a library that originate from the same cell?

The reverse transcription primer in the 10X Genomics Single Cell 3’ Gene Expression Library Prep Kit is composed of four distinct functional domains:

1. A 30 nucleotide oligo dT sequence
2. A 12 nucleotide unique molecular identifier
3. A 16 nucleotide 10X Cell Barcode
4. An Illumina Read 1 sequencing primer domain

A total of 3.5 million unique 10X Cell Barcodes are available with the 10X Genomics Chromium Single Cell 3’ Library and Gel Bead Kit v3. During cell partitioning, each cell is combined with a single gel bead that is loaded with reverse transcription primers that include one of the 10X Barcode sequences. These primers are used to reverse transcribe mRNA molecules that are released within the partition following cell lysis. The 10X barcode therefore marks the newly synthesized cDNA with a unique 16 nucleotide sequence that is common to all cDNA molecules localized within the same partition. Following sequence analysis, sequence reads sharing the same 10X barcode are implied to have originated from the same partitioned cell.

21. How many sequence reads are recommended for 10X Genomics 3’ Gene Expression libraries?

A minimum of 20,000 read-pairs per cell are recommended for libraries constructed with the 10X Genomics Chromium Single Cell 3’ GEM Library and Gel Bead Kit v3. Different cell types will require varying levels of sequence depth coverage. The sequencing saturation metric report that is generated from the Cell Ranger run summary can be used to optimize sequencing depth for individual cell types.

22. What does the term “Sequence Saturation” mean?

A 10X Genomics sequencing library consists of a collection of adapter-modified cDNA fragments that have been PCR amplified. Included within the adapter sequence of each amplified molecule is a unique molecular identifier (UMI) that is comprised of 12 random nucleotides that is added to each cDNA molecule during reverse transcription. Therefore, the UMI sequence is used to distinctly mark each cDNA molecule with a unique, random 12 base sequence. During sequence analysis, the UMI sequence enables the analyst to distinguish sequence reads derived from a unique cDNA molecule from those which consist of PCR replicates of the same cDNA molecule. The collection of UMI sequences that are observed in a data set combined with the frequency in which duplicate UMI sequences appear allows the Sequencing Saturation metric to be calculated. This measurement estimates the percentage of unique cDNA fragments within a library that are represented by sequence reads.

Therefore, 50% of sequencing saturation implies that approximately 50% of the complexity of the library has been sequenced. Additional sequencing of this library would initially present one new cDNA fragment (containing a novel UMI) for every two sequence reads acquired.

In contrast, deeper sequencing of a library that has achieved only 10% sequencing saturation will present nine new cDNA fragments for every ten additional reads acquired.

23. Are FACS sorted cells compatible with 10X Genomics single cell experiments?

FACS sorted cells are compatible with 10X Genomics single cell 3’ gene expression experiments. However, the cDNA yield from these samples is often lower than cells prepared by other methods. Prior to cell sorting, communicate with the Flow Cytometry Core Facility of your intention to use the cells for single cell sequencing experiments and discuss options that can reduce cell stress.

24. Are methanol-fixed cells compatible with 10X Genomics single cell gene expression library preparation?

Methanol fixation is compatible with many, but not all, cell types that are used with the 10X Genomics 3’ gene expression library preparation kits. A demonstrated protocol that describes methods for methanol fixation, storing, rehydrating, and recovering fixed cells for gene expression analysis can be assessed on the 10X Genomics website.

25. How do 3’ gene expression libraries differ from 5’ gene expression libraries?

Following cell partitioning, reverse transcription is initiated within emulsion droplets regardless of whether one is constructing a 3’ or a 5’ gene expression library. In the case of a 3’ gene expression library, a reverse transcription primer is released from a gel bead that is co-localized within a partition shared with a cell. This primer includes a 30 ntd oligo(dT) sequence, a 12 ntd UMI, a 16 ntd 10X barcode, and Illumina p5 adapter sequences. Reverse transcription of an mRNA using the 10X Genomics Single Cell 3’ Reagent Kit therefore results in the Illumina p5 adapter being directly attached to sequences representing the 3’ end of an mRNA molecule.

In contrast, reverse transcription with the 5’ gene expression library prep kit is primed with a standard oligo(dT) primer. On reaching the 5’ end of the mRNA molecule, the reverse transcriptase enzyme adds a short stretch of non-templated nucleotides to the cDNA. This non-templated sequence hybridizes to a 10X Genomics switch oligo that is released from a gel bead that is co-localized within a partition shared with the cell. The switch oligo, which also includes a 12 base UMI, a 14 base 10X barcode, and p5 Illumina adapter sequences enables reverse transcriptase to replicate these sequences by extending the 3’ end of the cDNA molecule. Therefore, when using the 10X Genomics Single Cell 5’ Reagent Kit, the Illumina p5 adapter sequence is associated with sequences complimentary to 5’ ends of mRNA transcripts.

26. Can cDNA from my 3’ Gene Expression library be used to construct a V(D)J library?

V(D)J libraries are constructed using cDNA that is generated with the 10X Genomics Chromium Single Cell 5’ Library and Gel Bead Kit. The 3’ gene expression library prep kit is not compatible with those reagents used to construct a V(D)J library.

27. What is the average insert size of a Single Cell 3’ Gene Expression Library?

The insert size of a library constructed with the 10X Genomics Chromium Single Cell 3’ Library and Gel Bead Kit v3 ranges from 250 bp to 550 bp with a mean insert size of approximately 350 bp.

28. How does the HTG Shared Resource qualify libraries prior to sequencing?

Quality control assays are performed to validate libraries prior to sequence analysis on the NovaSeq, HiSeq 2500, and MiSeq instruments. These assays include the following: Qubit dsDNA High Sensitivity Assay (library concentration), Agilent ScreenTape Assay (size distribution), and qPCR with the Kapa Library Quantification Kit for Illumina Platforms (normalize library representation in preparation for pooling). The cost for these quality control assays is included as part of library preparation when the HTG Shared Resource constructs the library. Alternatively, an additional fee is charged per sample when researchers construct libraries within their own lab.

29. How long will sequencing data from by genomic DNA libraries be available for download on the GNomEx server?

Sequencing data will be available on the GNomEx server for a period of approximately 6 months. The Bioinformatics Shared Resource has enabled an option for University of Utah laboratories to migrate sequencing data to Seven Bridges for long-term storage. Please contact the Bioinformatics Shared Resource for information on creating an account on Seven Bridges. Otherwise, researchers can explore other options for data storage but they should be aware that the GNomEx server is unable to support a solution in excess of 6 months.

30. Does the HTG Shared Resource provide assistance with analysis of sequence data?

The HTG Shared Resource does not provide sequence analysis services. Please contact the Bioinformatics Shared Resource (bioinformaticshelp@bio.hci.utah.edu) for assistance.