BioMicroCenter:SingleCell

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HOME -- SEQUENCING -- LIBRARY PREP -- HIGH-THROUGHPUT -- COMPUTING -- OTHER TECHNOLOGY

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.

10x CHROMIUM X

scRNA snATACseq multiome INPUT ~500-10,000 Cells or Nuclei Per Sample (CNPS) for NextGEM ~500-20,000 CNPS for GEM-X HT ~2,000-60,000 Cells or Nuclei Per Channel for HT FRP or probe based available for some assays, otherwise freshly counted cells preferred RECOMMENDED SEQUENCING NextSeq - 75nt kit 2-4 samples/flowcell NextSeq - 150nt kit 2-4 samples/flowcell NextSeq - 150nt kit 1-2 samples/flowcell SUBMISSION ASSISTED - ilabs WALKUP - Calendar DELIVERY FASTQ, SAM, BAM, 10X QC, loupe file UNIT PER SAMPLE or PER USAGE DONATED BY Prof. Manolis Kellis EXAMPLE DATA Example of QC Data from a 10x 3' v4 GEM-X assay. 10x Chromium X

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, 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. 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. The full 10x suite of software is installed on LURIA and is integrated into our analysis package. FAQs for Users 1) What buffers are compatible with 10X applications? Buffers/media should not contain excessive amounts of EDTA (>0.1 mM) or magnesium (>3 mM) and should be free of surfactants (i.e. Tween-20, SDS etc) and any RNases or DNases. 1xPBS (calcium free and magnesium free) containing 0.04% weight/volume BSA (400 µg/ml) is recommended for most general protocols and is considered the standard buffer Cell culture media with up to 1% BSA or up to 10% FBS if cells are not viable in standard buffer 1xPBS (calcium free and magnesium free) containing up to 10% FBS for CMO labelling Nuclei also require addition of RNase Inhibitor along with 10X Genomics 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 for pick-up. 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. 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. For more resources, please visit https://kb.10xgenomics.com/hc/en-us

NAMOCELL SINGLE CELL SORTER

INSTRUMENT NAMOCELL SINGLE CELL SORTER TYPE WALKUP - MIT only 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 ILABS NEW USERS New users should request training by emailing biomicro@mit.edu DONATED BY Prof Linda Griffith

The 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 integrates well with the TTP Labtech Mosquito HV which handles small reaction volumes. Namo (Namocell) Bio-Techne