Editing BioMicroCenter:SingleCell

{{BioMicroCenter}}

The BioMicro Center supports two technologies for single cell experiments: 10X libraries as walkup or as an assisted service, and plate based methods using the Namocell single cell sorter. <BR><BR>

<!-- commenting out Seq-Well 
== SEQ-WELL ==
{|
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  !   
  !SEQ-WELL 
  |-
  |INPUT
  |
* FULL PREP: <BR>50,000 single free cells. 90% viability
* EXO1 and AMP <BR> Tubes with cDNA attached to beads
* LIBRARY ONLY <BR> Amplified cDNA
  |-
  |THROUGHPUT 
  |
* FULL PREP/EXO: 4 samples/day
* LIBRARY: 24 samples/day
  |-
  |INCLUDED || QC at cDNA stage, library generation.
  |-
  |RECOMMENDED SEQUENCING || NextSeq500 - 75nt kit - 2-4 samples/flowcell
  |-
  |SUBMISSION || MIT - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=12903 ilabs] <BR> External contact biomicro@mit.edu
  |-
  |DELIVERY || FASTQ, SAM, BAM, Cell:Gene matrix
  |-
  |UNIT || PER ARRAY
 |}
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The BioMicro Center collaborates closely with the Nanowell Core in the Koch Institute to provide support for the [https://shaleklab.com/resources/seq-well/ Seq-Well] protocol developed by Drs. Alex Shalek and Chris Love's groups. Seq-Well is based on the isolation of individual cells into microwells where cells are lysed and the associated mRNA is attached to polydT containing beads.  <BR><BR>
Seq-Well projects can be brought in two ways. For full service, please use the ilabs form linked on the left. At the end of the form it asks you to select several days on which the experiment can be done. The Nanowell Core will review the possible dates with you to fit it in the schedule. On that date, the core will confirm the quality of your cells prior to beginning the experiment. While 10,000 cells are typically used in a Seq-Well experiment, the core requests you plan to bring more cells - often counts from flow cytometers and other instruments are incorrect. Planning to bring more cells ensures you have the 10,000 desired. The BioMicro Center becomes involved in these projects at the exonuclease treatment step, where we take over the preparation of the library and quality control, as well as sequencing and analysis.<BR><BR>
Many laboratories are also using reagents from the Nanowell core to begin doing Seq-Well on their own. For these labs, we strongly recommend at least confirming the quality of the cDNA on our Advanced Analytical before continuing through library preparation. The library preparation can also be done in the BioMicro Center. Please use the standard Illumina library preparation form and note that the samples are Seq-Well. We do have all the custom primers needed for preparation and the pricing is identical to other NexteraXT preps. 

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-->
== 10x CHROMIUM X ==
{|
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  !   
  !scRNA
  !snATACseq
  !multiome
  |-
  !INPUT 
  |colspan="3"| ~500-10,000 Cells or Nuclei Per Sample (CNPS) for NextGEM <BR> ~500-20,000 CNPS for GEM-X <BR> HT ~2,000-60,000 Cells or Nuclei Per Channel for HT <BR> FRP or probe based available for some assays, otherwise freshly counted cells preferred
  |-
  !RECOMMENDED SEQUENCING 
  |NextSeq - 75nt kit <BR>2-4 samples/flowcell
  |NextSeq - 150nt kit <BR> 2-4 samples/flowcell
  |NextSeq - 150nt kit <BR> 1-2 samples/flowcell
  |-
  !SUBMISSION 
  |colspan="3"| ASSISTED - [https://mit.ilabsolutions.com/service_item/new/3381?spt_id=12903 ilabs] <BR> WALKUP - [https://mit.ilabsolutions.com/equipment/380963 Calendar]
  |-
  !DELIVERY 
  |colspan="3"| FASTQ, SAM, BAM, 10X QC, loupe file
  |-
  !UNIT 
  |colspan="3"| PER SAMPLE or PER USAGE
  |-
  !DONATED BY 
  |colspan="3"| [http://mit.edu/manoli/ Prof. Manolis Kellis]
  |-
  !EXAMPLE DATA
  |colspan="3"| [[image:BMC_10X_data.jpg|thumb|left|600px|Example of QC Data from a 10x 3' v4 GEM-X assay.]]
 |}
 |

The BioMicro Center provides access to 10x Genomics library preparation as an assisted or a walk-up service. Added in 2023, The 10x Chromium X can handle a broad variety of methodologies now including [https://www.10xgenomics.com/solutions/single-cell/ 3' and 5' RNA sequencing], [https://www.10xgenomics.com/solutions/single-cell-atac/ ATACseq] and [https://www.10xgenomics.com/solutions/single-cell-cnv/ CNV]. For users requesting assisted service, they will bring their single cell suspension to the core at a time coordinated with the center staff. We will then work with you to get the initial samples loaded and proceed through the protocol with a quality control check at the amplified cDNA state. 
<BR><BR>
This is also offered as a walkup service. Usage may be scheduled on the ilabs calendar after training by BMC staff. You will not have permission to schedule the equipment until you have been approved by BMC staff. Reagents are stocked in the BioMicro Center and we ask that you use our reagents. This allows us to get larger bulk discounts we can pass on to our users. Please note that we do include a fraction of the instrument usage cost in the cost of the chips and will charge this cost if you use your own chips.
<BR><BR>
The full 10x suite of software is installed on LURIA and is integrated into our analysis package. <BR><BR>

FAQs for Users <BR>
1) What buffers are compatible with 10X applications?
- It is recommended to use 1X PBS (calcium and magnesium-free) containing 0.04% weight/volume BSA (400 µg/ml) for washing and resuspension of cells for 10x applications. Fresh and frozen/thawed PBMC samples and cell lines have been tested with this buffer. However, for Multi-ome and ATAC applications, nuclei must be suspended in 1X nuclei buffer before chip loading, instructions for which are included in 10x user guides. If needed, users can collect buffer aliquots from the BMC after submitting a project. Please coordinate with BMC staff to set it up. <br>
2) How many cells are captured in the Assay?
-Up to 10,000 cells for NextGEM and 20,000 cells for GEM-X can be uniquely barcoded, but this highly depends on cell counts and viability. Dying cells will leak RNA, hence may not be captured efficiently leading to sample failures. We recommend to count cells at the BMC to avoid discrepancies, but can work with users' counts as well. <BR>
3) What are the best practices for flow sorting cells?
-https://kb.10xgenomics.com/hc/en-us/articles/360048826911-What-are-the-best-practices-for-flow-sorting-cells-for-10x-Genomics-assays <BR>
4) What is the expected size distribution for cDNA?
- cDNA for 3' and 5' libraries will span between 400 to 9000 base pairs, depending on sample type.

For more resources, please visit https://kb.10xgenomics.com/hc/en-us
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|}

== NAMOCELL SINGLE CELL SORTER ==

{| align=left 
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  ! INSTRUMENT|| NAMOCELL SINGLE CELL SORTER
  |-
  | TYPE || WALKUP - MIT only <BR> FACS
  |-
  | UNIT || Per Cartridge
  |-
  | LASER WAVELENGTH	||  488 nm
  |-
  | DETECTION CHANNELS
  |
* FL1 533 nm (FITC/GFP)
* FL2 585 nm (PE/PI)
* FL3 676 nm (PerCP)
  |-
  | DISPENSE VOLUME || 1 ul
  |-
  | SAMPLE VOLUME || 100-750 ul
  |-
  | CELL INPUT || single cell mode: 100-10,000 cells 
  |-
  | FORMAT || 96w/384w
  |-
  | SIGNUP || [https://mit.ilabsolutions.com/equipment/show/370751/?tab=schedule ILABS]
  |-
  | NEW USERS ||  New users should request training by emailing biomicro@mit.edu
  |-
  | DONATED BY || Prof Linda Griffith
 |}
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The [https://www.namocell.com/namo/ Namocell Single Cell Sorter] allows users to sort cells into plates. The sorter uses microfluidics to sort single cells in 1uL of sheath fluid into a well. The instrument uses disposable cartridges to minimize contamination. The instrument is expected to integrate well with the [[BioMicroCenter:Tecan_Freedom_Evo#TTP_LABTECH_MOSQUITO_HV|TTP Labtech Mosquito HV]] which handles small reaction volumes. <BR><BR>
[[image:Namo.jpg|thumb|right]]
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|} 

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