CRUK-CI Genomics Help

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submission:10x_submissions [2025/02/13 17:51] – [10x sequencing configuration recommendations] Bioinformatics service adminsubmission:10x_submissions [2026/04/10 12:57] (current) – [Single Cell Submission] Johanna Barbieri
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 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]]\\ 
 +__**It is recommended for any single cell 10x Genomics project to consider a pilot experiment prior to a multi-sample project**__
  
 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. \\ 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. \\
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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| |10x Multiome|Next GEM Multiome|Nuclei| 5|3200|10|16,000|10,000|0.2ml tubes|
  
-And for the 10x Genomics GEMX Flex workflows:+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 ^ ^Workflow ^Kit used ^ Sample type ^Max Number of Cells to Submit ^Max  # Cells Loaded ^ # Cells Recovered ^ Preferred Eppendorf ^
-|10x Single Cell GEMX Flex|GEM-X Flex Singleplex|Cells/Nuclei|300,000|29,250 |20,000 |1.5ml tubes| +|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|GEM-X Flex Multiplex|Cells/Nuclei|300,000|116,000|80,000 |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.  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. 
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 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.  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. 
  
-Parse similarly suffers from poor sample fixation and counting which necessitates these steps having been done with the utmost care. Failures with Parse are not as immediately obvious as 10x Genomics such that we can stop the sequencing to ensure no further wasted funds. +
 ===== Cell Suspension Advice ===== ===== Cell Suspension Advice =====
  
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 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. 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. +__**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.