Single Cell Submission

This section covers chemistries including 10x Genomics - 3'/5' TCR/BCR Cell-Surface Protein expression, CRISPR, and enrichment strategies e.g WILD-seq, Total-seq etc and Parse WT - Mini/Midi/Mega.

Due to the nature of processing single cell samples, for all services you must discuss your project with the Genomics Core prior to submission. We cannot offer a walk-up service or accept samples that are not discussed in advance.

In the first instance, contact the single cell team to discuss details of the cells or nuclei submission to ensure they are aware of the experimental conditions and timelines.
Single cell team

For 10x Genomics, We ask you to bring your cell suspension or isolated nuclei in the following required concentration in 1x PBS +0.04% BSA. Alternatively, there are other buffers acceptable that 10x Genomics list on their website found here: https://kb.10xgenomics.com/hc/en-us/articles/115001937123-What-buffers-can-be-used-for-washing-and-cell-resuspension.
Below are our sample submission requirements & based on standard 10X Genomics recommendations:

And for the 10x Genomics GEMX Flex workflows - fixed samples (freshly fixed, formalin-fixed paraffin embedded (FFPE)):

10x Genomics recommend a minimum viability of 70% but sample preparation is the key step in yielding good single-cell resolution data. The system is optimised to work with high quality cell/ nuclei input with accurately calculated counts. Any degradation in viability & quality or inaccurate counting is likely to end with sub-optimal results and potentially useless data.

Almost all failures are due to poor quality sample preparation in excess debris/ underestimated cell counts/ unsupported buffers for suspension in the cell or nuclei suspension we receive. We do not quality check or count cells for you unless required by the protocol (GEMX Flex) and even then we ask you/ a member of your lab to be present to ensure we can proceed confidently.

As mentioned above its to optimise your dissociation and counting methods for your particular cell sample, please contact the single cell team or visit the 10X website to find guidance on how best to optimise your protocols prior to submitting your samples to our team. https://www.10xgenomics.com/support/universal-three-prime-gene-expression/documentation/steps/sample-prep/tumor-dissociation-for-single-cell-rna-sequencing

For Parse, as previously stated, the only additional advice would be to have fixed a counting aliquot alongside your main sample allowing you to count the day before and assess quality. Makes submission on the day easier and quicker as we require submission before 11am.

Samples should be submitted in a clearly labelled tube (see table above) and brought on ice.

It is recommended for any single cell 10x Genomics project to consider a pilot experiment prior to a multi-sample project. This may not be possible for Parse given the wholistic use of the kit but potentially utilising their Mini kit first may alleviate significant investment waste. For any other questions you might have about cell preparation, counting and sequencing please see our FAQ page.

The first few fields are related to which workflow you want your samples to go through. We ask for submission forms to be completed and submitted within 2 days of your submission regardless of if you have more samples to submit in the near future.

• Workflow: Please choose from the drop-down menu the experiment type you want processed.
• Additional workflows: Can be left blank if additional workflows are not needed. In the drop-down menu is our most common additional workflows for example if you need CITE-Seq/Cell surface Protein information. All additional Workflows need to be discussed with us in advance, please contact us to discuss.
• VDJ Amplification: Only relevant if you choose “10x Single Cell V(D)J” in the workflow field. Do you want TCR, BCR or both enrichments?
• Ready to sequence?
  • No: if you are still submitting samples that you want pooled and sequenced together. You do not need to fill in the sequencing information (sequencer, flow cell type, number of lanes).
  • Yes: if you have submitted all your current samples and are ready to sequence. Please contact us and let us know which samples you want pooled. You will have to fill in the sequencing information (sequencer, flow cell type, number of lanes).

The following information is needed if your samples are ready to sequence, otherwise it can be left blank:

• Sequencer: For 10x Genomics workflows we recommend using NovaSeq X as it produces the best quality/ necessary number of reads.
• Flow Cell Type: There are three sizes of flow cells to be used on NovaSeq X, producing different number of reads per lane (see the table below). If you are unsure how many reads you need, see our FAQ page or contact us to seek advice. The table below gives a guide to the relative sizes.
• Number of lanes: Strictly connected to flow cell type and your decision about number of reads needed.

N.B The 1.5B and 10B flow cells can also come in different cycle kits i.e. 100 cycles/ 200 cycles/ 300 cycles but the 25B comes as 1 allowing us to run most of the different parameters (within reason) on the same 25B flow cell. These vary in price but can allow different workflows with different sequencing parameters to be sequenced on the same flow cell, NOT the same lane.

The following information is needed:

• Species: Information used only for our Multi-Genome Alignment report. If multiple different species used, please make a note in the submission comment section so we are not concerned that sample has been contaminated.
• Billing Information: For 10x Genomics library preparation, collaboration codes MUST NOT be used. Please contact us prior to your experiment to set up your grant code in the system.
• PO Number: Applicable only for commercial users. Leave blank if it does not apply! Resist the temptation to put “N/A” or “None”: it will complicate your bills.

Individual sample information:

• Name: please be consistent with what you label on your tube and what you fill on the submission sheet. Have either date and numbers or a continuous number system on tubes and filling in the form so it makes it easy for us to cross reference samples processed and submitted into our system E.g. if 3 samples are submitted on 3rd May:
  • Date and numbers use YYMMDD (Year Month Day with no spaces) in the same format:
    • 230503_1_Sample_Name
    • 230503_2_Sample_Name
    • 230503_3_Sample_Name
  • OR continuous number system
    • 1_sample_Name
    • 2_sample_Name
    • 3_sample_Name
• Concentration: Concentration of cell suspension provided to our facility in [cells/µl].
• Volume: Volume of cell suspension provided to our facility in [µl]. Please refer to table above: for example, 55µl should be provided when submitting for 3' v3.1 workflow- we will load 43.2µl.
• Species: Only necessary if different to value in cell C17.
• Reads per cell: This field is optional, but it will help us in advising you on which flow cell to use, and if we notice any inconsistencies.
  

Please see below for 10x Genomics recommendations for number of reads per workflow but we are happy to pool and sequence as you request. We require written confirmation to proceed with alterations to standard procedures but will most likely email you just to double check.

• Total-Seq B/C are 5k reads per cell as are most enrichments but as previously stated, happy to modulate upon request.
• Submission comments: Anything that needs to be communicated and might be relevant for the experiment like suggestions about pooling before sequencing, type of antibodies used for staining (CITE-Seq), information about possible low RNA content etc.