The BioMicro Center supports three technologies for single cell experiments: SeqWell, in collaboration with the Koch Institute FlowCytometry:Nanowell core, 10X libraries as walkup or as an assisted service, and plate based methods using the Namocell single cell sorter.
SEQWELL
SEQWELL 3'DGE
INPUT
FULL PREP: 50,000 single free cells. 90% viability
The BioMicro Center collaborates closely with the Nanowell Core in the Koch Institute to provide support for the SeqWell protocol developed by Drs. Alex Shalek and Chris Love's groups. Seqwell is based on the isolation of individual cells into microwells where cells are lysed and the associated mRNA is attached to polyT containing beads.
Seqwell 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 SeqWell 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.
Many laboratories are also using reagents from the Nanowell core to begin doing SeqWell 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 SeqWell. We do have all the custom primers needed for preparation and the pricing is identical to other NexteraXT preps.
10X CHROMIUM X
scRNA
snATACseq
scCNV
INPUT
~10,000 Cells for NextGEM ~20,000 for GEM-X fresh cultures <~30' old
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 3' and 5' RNA sequencing, ATACseq and CNV. For users requesting assisted service 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.
Many users may also wish to use the system 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.
One of 10X's strengths is their software package for analysis. The full 10X suite is installed on LURIA and is integrated into our analysis package.
FAQs for Users
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, instructions for which are included in the 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.
2) How many cells are captured in the Assay?
-Upto 10,000 cells 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.
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
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.
New users should request training by emailing biomicro@mit.edu
DONATED BY
Prof Linda Griffith
The Namocell Single Cell Sorter is a new instrument in the BioMicro Center that 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 TTP Labtech Mosquito HV which handles small reaction volumes.
