CRUK-CI Genomics Help

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submission:10x_submissions [2025/02/13 17:46] – [How to Complete the 10x Submission Form] Bioinformatics service adminsubmission:10x_submissions [2025/02/14 11:41] (current) – [Single Cell Submission] Ania Piskorz
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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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   * **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.