CRUK-CI Genomics Help

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submission:10x_submissions [2025/02/13 15:08] – [Single Cell 10x Genomics Submission] Bioinformatics service adminsubmission:10x_submissions [2025/02/14 11:41] (current) – [Single Cell Submission] Ania Piskorz
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-====== Single Cell 10x Genomics Submission ====== +====== Single Cell Submission ====== 
-This section covers chemistries including 3'/5' TCR/BCR Cell-Surface Protein expression, CRISPR, and enrichment strategies e.g WILD-seq, Total-seq etc.\\ +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 10x Genomics 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.\\+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.\\ 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.\\
 [[singlecell@cruk.cam.ac.uk|Single cell team]] [[singlecell@cruk.cam.ac.uk|Single cell team]]
  
-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. \\ +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.+Below are our sample submission requirements & based on standard 10X Genomics recommendations:
  
 ^Workflow ^Kit used ^ Sample type ^ Volume loaded (µl) ^Max Target Concentration (cells/µl) ^Volume for User to Submit (µl) ^ # Cells Loaded ^ # Cells Recovered ^ Preferred Eppendorf ^ ^Workflow ^Kit used ^ Sample type ^ Volume loaded (µl) ^Max Target Concentration (cells/µl) ^Volume for User to Submit (µl) ^ # Cells Loaded ^ # Cells Recovered ^ Preferred Eppendorf ^
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 |10x Single Cell 5GEX|Next GEM 5'v2|Cells/Nuclei|39.1|409|45|16,000|10,000|1.5ml tubes| |10x Single Cell 5GEX|Next GEM 5'v2|Cells/Nuclei|39.1|409|45|16,000|10,000|1.5ml tubes|
 |10x Single Cell 5GEMX|GEM-X 5'v3|Cells/Nuclei|37.5|667|45|25,000|20,000|1.5ml tubes| |10x Single Cell 5GEMX|GEM-X 5'v3|Cells/Nuclei|37.5|667|45|25,000|20,000|1.5ml tubes|
-|10x Single Cell 3' v4 4-Plex|GEM-X 3' OCM|Cells/Nuclei|9.6|860|15|8,250|5,000|1.5ml tubes| +|10x Single Cell 3GEMX OCM 4-plex|GEM-X 3' OCM|Cells/Nuclei|9.6|860|15|8,250|5,000|1.5ml tubes| 
-|10x Single Cell 5' v4 4-Plex|GEM-X 5' OCM|Cells/Nuclei|9.8|842|15|8,250|5,000|1.5ml tubes|+|10x Single Cell 5GEMX OCM 4-plex|GEM-X 5' OCM|Cells/Nuclei|9.8|842|15|8,250|5,000|1.5ml tubes| 
 +|10x Multiome|Next GEM Multiome|Nuclei| 5|3200|10|16,000|10,000|0.2ml tubes|
  
 +And for the 10x Genomics GEMX Flex workflows - fixed samples (freshly fixed, formalin-fixed paraffin embedded (FFPE)):
 +
 +^Workflow ^Kit used ^ Sample type ^Max Number of Cells to Submit ^Max  # Cells Loaded ^ # Cells Recovered ^ Preferred Eppendorf ^
 +|10x Single Cell GEMX Flex Singleplex 1plex 4 samples|GEMX Flex Singleplex|Cells 1plex/Nuclei|300,000|29,250 |20,000 |1.5ml tubes|
 +|10x Single Cell GEMX Flex Multiplex 4plex 16 samples|GEMX Flex Multiplex|Cells 4plex/Nuclei|300,000|116,000|80,000 |1.5ml tubes|
      
 +For Parse, we ask you to utilise an excel sheet that Parse provide to determine both concentration and volume to target for your submissions to us, this includes a dilution step upon our receipt before loading to the first barcoding plate. These excel sheets can be found on Parse's website under the kit you used (Mini/Midi/Mega) as they are different for each. Additionally, we would highly recommend to aliquot part of your samples to count the day before submission without freeze-thawing and allowing you to drop samples off earlier on the day. 
 +
 ===== Sample Preparation ===== ===== Sample Preparation =====
-10x Genomics recommend a minimum viability of 70% but sample preparation is the key step in yielded 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.+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. +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. 
  
  
-===== Cell suspensions advice =====+===== Cell Suspension Advice =====
  
 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. 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 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. 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.+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 [[https://genomicsequencing.cruk.cam.ac.uk/sequencing/help/Help!tenxFAQ.action|FAQ]] page. For any other questions you might have about cell preparation, counting and sequencing please see our [[https://genomicsequencing.cruk.cam.ac.uk/sequencing/help/Help!tenxFAQ.action|FAQ]] page.
  
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 ^Flow Cell Type NovaSeq X ^Number of lanes ^Specification in Billion Reads per lane^ ^Flow Cell Type NovaSeq X ^Number of lanes ^Specification in Billion Reads per lane^
-|  1.5B   |   2  |   0.75  |+|  1.5B   |   2  |   0.80  |
 |  10B  |   8  |  1.25  | |  10B  |   8  |  1.25  |
 |  25B  |   8  |   3.125  | |  25B  |   8  |   3.125  |
-N.B These flow cells can also come in different cycle kits i.e 100 cycles/ 200 cycles/ 300 cycles. 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.+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: 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.   * **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.+  * **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.   * **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.
  
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   * **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.\\   * **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.\\
  
-===== 10x sequencing configuration recommendations ===== +===== 10x Sequencing Configuration Recommendations ===== 
-10x Genomics have recommendations for number of reads per workflow: +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.
  
 ^Single Cell Chemistry ^Feature ^Configuration:\\ Read1/i7/i5/Read2 ^  Sequencing Depth  ^ ^Single Cell Chemistry ^Feature ^Configuration:\\ Read1/i7/i5/Read2 ^  Sequencing Depth  ^
-|Single Cell 3'v4/v3.1/Dual index | Gene Expression|  28/10/10/90  |     20K read pairs / cell    |+|Single Cell 3'v4/3'v3.1/OCM/Dual index | Gene Expression|  28/10/10/90  |     20K read pairs / cell    |
 |:::| FB CRISPR|:::|  5K read pairs / cell  | |:::| FB CRISPR|:::|  5K read pairs / cell  |
 |:::| FB Cell Plex|:::|  5K read pairs / cell  | |:::| FB Cell Plex|:::|  5K read pairs / cell  |
 |:::| FB Cell Surface Protein|:::|  5K read pairs / cell   | |:::| FB Cell Surface Protein|:::|  5K read pairs / cell   |
-|Single Cell 5'v3/5'HT/Dual index | Gene Expression|  28/10/10/90  |  20K read pairs / cell  |+|Single Cell 5'v3/OCM/Dual index | Gene Expression|  __28__/10/10/90  |  20K read pairs / cell  |
 |:::| V(D)J|:::|  5K read pairs / cell  | |:::| V(D)J|:::|  5K read pairs / cell  |
 |:::| FB CRISPR|:::|  5K read pairs / cell  | |:::| FB CRISPR|:::|  5K read pairs / cell  |
 |:::| FB Cell Surface Protein|:::|  5K read pairs / cell  | |:::| FB Cell Surface Protein|:::|  5K read pairs / cell  |
-|Single Cell 5'v2/Dual index | Gene Expression|  26/10/10/90  |  20K read pairs / cell  |+|Single Cell 5'v2/Dual index | Gene Expression|  __26__/10/10/90  |  20K read pairs / cell  |
 |:::| V(D)J|:::|  5K  read pairs / cell   | |:::| V(D)J|:::|  5K  read pairs / cell   |
 |:::| FB CRISPR|:::|  5K read pairs / cell  | |:::| FB CRISPR|:::|  5K read pairs / cell  |
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 |Single Cell Multiome | ATAC|  50/8/24/49  |  25K read pairs / cell  | |Single Cell Multiome | ATAC|  50/8/24/49  |  25K read pairs / cell  |
 |:::| GEX|  28/10/10/90  |  20K read pairs / cell  | |:::| GEX|  28/10/10/90  |  20K read pairs / cell  |
-|Flex (Fixed RNA Profiling) | Singleplex|  28/10/10/90  |  10K read pairs / cell  |+|GEMX Flex (Fixed RNA Profiling) | Singleplex|  28/10/10/90  |  10K read pairs / cell  |
 |:::|Multiplex |:::|  10K read pairs / cell  | |:::|Multiplex |:::|  10K read pairs / cell  |
 |:::|Protein |:::|  5K read pairs / cell  | |:::|Protein |:::|  5K read pairs / cell  |
  
-    * Total-Seq B/C are 5k reads per cell. +    * 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.   * 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.