BioMicroCenter:SingleCell

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

The BioMicro Center supports a broad range of methods for single cell sequencing. The choice of method depends heavily on the type of question being asked and the source material. The methods break down into those supporting BioMicroCenter:SingleCell#NAMOCELL_SINGLE_CELL_SORTER individual cells characterized in single wells, methods that BioMicroCenter:SingleCell&action=submit#10x_CHROMIUM_X use droplet isolation and library preparation, and those that support rapid characterization of a modest number of transcripts using in situ sequencing (coming soon). These cell requirements for each method vary significantly so reviewing each method is valuable and we strongly encourage consultation with BMC staff prior to beginning the experiment.





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10x CHROMIUM X

scRNAseq / snRNAseq 3'RNA/5'RNA (GEM-X) 3'/5' OnChipMultiplexing (OCM) Fixed RNA (Flex GEM-X) INPUT Freshly counted cells/nuclei in suspension. Fixed cells in suspension. nuclei/uL 100-2000 100-2000 2000+ Fraction Recovered est. percent of input cells captured ~65% ~60% ~50% Target recovery multiply by recovery for input 500-20,000/lane 500-5,000/lane 500-10,000/sample Expected doublet rate @ Max load 8% 7.5% 8% doublet @ 10k Multiplexing 1 sample 4 samples/library 4 samples/lane ADD'L AMPLICONS Custom priming for CITEseq, CRISPRs (perturb-seq), TCR (5'), BCR (5'). Please provide custom oligos and protocols to core. Additional genes require new probes. KEY NOTES Human/Mouse only SUBMISSION FORMS ASSISTED - ilabs WALKUP - Calendar DELIVERY FASTQ, SAM, BAM, 10X QC, loupe file DONATED BY Prof. Manolis Kellis CHROMATIN snATACseq multiome Input 160-8,000 nuclei/uL 50% estimated recovery 7.5% doublets @ 10k cells recovered 10uL minimum volume Multiplexing Not available SUBMISSION FORMS ASSISTED - ilabs WALKUP - Calendar DELIVERY FASTQ, BAM, 10X QC, loupe file

10X Genomics 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', 5' and fixed RNA sequencing, ATAC and multiome Dropoff for assisted service is coordinated with Center staff with at least one week's lead time. Users should bring their single cell/nuclei suspension(s) in 1.5 mL microfuge tubes in the standard buffer at or around that time. Staff works with the user to intake the initial samples and proceed through the protocol with quality control checks at the appropriate steps. Chromium X usage is also offered as a walkup service. Usage may be scheduled on the iLabs calendar after training by BMC staff. This still requires at least a week's lead time before usage. 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 in the final suspension are compatible with 10X applications? Buffers/media for the submitted single cell/nuclei suspension 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? 10x provides guidance with their tested protocols about pre-sort buffer, collection buffer and FACS best practices here. 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. 5) How much sequencing per sample is recommended? 10x makes several recommendations in their sequencing handbook. Recommendation numbers vary by sample type, expected CNPs per sample, assay type and general sample quality. The higher the CNPs, the higher the quality, the more likely increased read depth is required. 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