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Single-Cell RNA-Sequencing Pipeline

  • CELL INPUT/EXPERIMENTAL DESIGN – Core personnel will conduct an initial consultation to discuss sample specific details of your study and to help begin planning the timeline of the experiment
  • CELL SORTING – Cells can be sorted to isolate viable populations free of debris to be run through the 10X controller for best results
  • 10X GENOMICS – Purified samples are run through 10X controller and individually bar-coded libraries are generated and pooled
  • RNA SEQUENCING – In partnership with the Molecular Biology Core Facilities (MBCF) at Dana-Farber Cancer Institute (DFCI), pooled library samples are sequenced
  • BIOINFORMATIC ANALYSIS – Raw data are processed using Cell Ranger and standard QC to generate count matrices and browseable Loupe Cell data files.

GENERAL

What is the cost per sample?

All the information about pricing is listed under the Services & Pricing menu. In brief, it depends on how many samples you commit to schedule within a 3-month period:

  • 1-9 samples: $4250/sample
  • >10 samples: $3500/sample
  • We also offer Cell Hashing, Cite-Seq and Immune Cell Repertoire services provided at an ‘add-on’ price. For example, if you multiplex several samples using cell hashing technology, the price will be $4250 (base price for 1 pooled set of cell populations) + $2000 (‘add-on’ price for the Cell Hashing service).

What is included in the price?

We provide a full, all-inclusive, comprehensive service which includes:

  • Administrative services, consultations and advice on experimental design and sample preparation
  • All 10x Genomics reagents, buffers, and Cell Hashing antibodies (if required)
  • Sample QC prior to loading, and post-library generation
  • Sequencing and basic bioinformatic analysis (using the Cell Ranger pipeline)
  • Trouble-shooting assistance and re-sequencing, if necessary, to aim to achieve at least 20k reads/cell

How should I book the experiment?

    1. Create an account on the Partners Core Management System
    2. If you are a new user, or this is a new experiment, please fill out our Questionnaire and send to SingleCell@bwh.harvard.edu
    3. Our scientific team will review your Questionnaire and set up a consultation to discuss your experiment
    4. Upon completion of the consultation:
    5. A core representative will respond to you to schedule your sample submission
    6. All samples must be submitted to the core by no later than 2pm, Monday – Friday

I want my data as soon as possible! What is the Turn Around Time for your core?

It takes about 2 months, from the time the sample is submitted, to data delivery after running the CellRanger pipeline.

CELL SORTING

Why is it strongly suggested to sort cells prior to use of the 10X platform?

Sorting for viable cells that are purified from any debris or contaminating cells increases the effectiveness of the 10X controller and helps to avoid detrimental clogs as well as produces significantly better sequencing and data analyses downstream.

Why sort at the Center for Cellular Profiling?

The Center for Cellular Profiling: Cytometry Facility is located in the same laboratory as the Single-Cell Multiomics Facility and it has been proven that decreasing the time between isolating viable cells and the use of the 10X controller produces significantly better results.

Is cell sorting included in the pricing package offered?

The first 2 hours of any cell sorting experiment specifically used for Single-Cell experiments is covered. Any additional sorting time will be charged based on our current fees listed below.

How much does is cost to sort?

Fees are as follows:

  • MGB Academic – $110/hour
  • Non-MGB Academic – $159/hour
  • For-Profit Industry/Biotech – $200/hour

How do I schedule a sort?

During your consultation with the Single-Cell Scientists, the core manager and scientific directors will help you coordinate a time that works for both facilities and that fits your schedule.

Where can I find more information on cell sorting?

Please navigate to the Center for Cellular Profiling Flow Cytometry Services menu for more information or contact: FACS@bwh.harvard.edu.

10X GENOMICS

How many samples should I run?

The number of samples should be determined based on your experimental goals.  We can support up to 8 samples each day, however it is highly recommended that the investigator perform a pilot experiment before committing to large scale experiment.

How many cells do I need to provide and how many cells can I expect to get information for?

We recommend loading between 10k live cells (for non-hashing experiments) and 20-30k live cells (for cell hashing experiments). Therefore, please provide at least double this cell number, so that there are enough cells to re-load the sample if a wetting failure occurs during the 10x encapsulation. The capture rate of 10x is approximately 60%, depending on cell type and cell quality.

How should the cells be prepared for 10X?

It is recommended that investigators should optimize their cell isolation procedure prior to 10X experiments. In general, we recommend cell viability of >85% for optimal cell input. The Center for Cellular Profiling: Flow Cytometry Sorting Facility can be used to isolate viable cells or there are protocols available for cell isolation by MACs beads. For single cell RNA-seq, we ask you to resuspend your cells in 0.4%BSA/PBS. We can provide this buffer, or it is fine to prepare it yourself using nuclease free, filtered reagents. For single nuclei RNA-seq, 1% BSA/PBS with 0.2U/μl RNase Inhibitor is required (we can provide the 1% BSA/PBS, but you will need to supply your own RNase Inhibitor). For ATAC-seq, we can provide the 20X Nuclei Buffer – please dilute 1:20 in nuclease-free water before use.

How many reads per cell can I expect?

We aim to provide at least 30k reads/cell for gene expression libraries, as recommended by 10x Genomics. For Cite-seq libraries, we provide a sequencing depth of 5,000 reads/cell. If you are using more than 100 TotalSeq™ antibodies, we will increase the read number to 10,000 reads/cell. For cell hashing libraries, we provide a sequencing depth of 5,000 reads/cell.

What are the advantages and disadvantages of cell hashing technology?

Using cell hashing technology, samples treated in different conditions can be pooled together, thereby decreasing batch effect. Also, it is cost-effective when pooling several samples. However, the main disadvantage of this technology is that fewer cells from the individual conditions can be loaded. For example, when loading 30k total cells in a cell hashing experiment, if you plan to pool 6 conditions in one 10x run there will be 5000 cells/condition, rather than the 10k cells we would normally load in a non-hashtag labelled sample. In addition, the doublet rate with 30k cell input will be much higher than 10k cells, so the number of cells passing QC will be less.

How do I know if my cell type is compatible with cell hashing technology?

To perform cell hashing, you need to make sure:

  1. Your cells can be labelled with hashtag antibodies. For human samples, the hashtags are made of two antibodies that recognize ubiquitous surface markers, CD298 and β2 microglobulin, each conjugated to the same oligonucleotide containing the barcode sequence. For mouse samples, the surface markers are CD45 and H-2 MHC class I. The conjugates are already pre-mixed and ready to use.
  2. Your cells can maintain a high viability after the labelling process. We can provide the protocol and relevant staining buffers to researchers to perform a ‘mock’ labelling experiment and obtain a final viability assessment. We require the viability of the submitted samples to be no less than 85%.

Does the core provide cell hashing antibodies?

Yes, we can provide the relevant cell hashtag antibodies, Fc blocking reagent, and staining buffer. After you book your experiment, please coordinate with us to pick up those reagents one day in advance.

Does the core provide Cite-seq antibodies?

No, the core does not provide Cite-seq antibodies. Please order the antibody panels from Biolegend yourself. Please confirm with us the Total-seq format you are planning to use (e.g. Totalseq A or B or C)

Do I need to pool the cells from different populations myself if I plan to do Cell hashing?

Yes. You will need to count the cell number of each population, pool to the required proportions (usually in equal numbers) prior to submitting the pooled sample to us. We will then perform a confirmatory assessment of the cell concentration and viability of the sample prior to running on the 10x Controller. However, if you are using our on-site FACSorting Facility (the CCP Flow Cytometry Facility) the individual post-sorted hash-tagged cell populations can be counted and pooled, per the client’s instruction, by us, if preferred.

RNA SEQUENCING

Does the cost of the package pricing include sequencing cost?

Yes.

Where are the libraries sequenced?

Sequencing is performed by the Molecular Biology Core Facilities (MBCF) at Dana-Farber Cancer Institute (DFCI).

What is your sequencing platform?

We use Illumina NovaSeq 6000 (S4, S2, S1 and SP flowcell), HiSeq X, and NextSeq 500 systems.

Do you pool libraries for sequencing?

Yes. The pooled number depends on the cell input of the libraries and the outputs of sequencing platform.

Will I be able to get the raw sequencing data?

Yes, we routinely deliver BAM files, which FASTQ files (raw data) can be obtained from by performing bamtofastq function within Cell Ranger software.

BIOINFORMATIC ANALYSIS

What does the core package include for analysis?

Data generated from the core will be processed through the standard Cell Ranger pipeline (10X Genomics). Raw data, QC metric data, count matrices, and browsable data files in Loupe Cell format will be made available to investigators.

What are the steps taken for QC?

The Cell Ranger pipeline performs initial QC and provides a matrix with counts per GEMs that are likely cells. We recommend performing extra-downstream filters based on number of genes per cell and mitochondrial content (appropriate thresholds may vary per experimental design and cell type).

How much time will it take to get my data?

The timeline will be based on sample numbers and the scheduling queue of the core.

Is there an option for additional analysis beyond what is offered in the package?

Unfortunately, at this time, we are unable to provide this service. We hope to soon!

How can I understand the data output and get recommendations of downstream analyses?

You can check out our Data Output User Guide, which explains the folder structure of the data, and provides links to tutorials, packages, books and articles that can help you analyze the data.

Are there any groups at BWH that meet to share knowledge and skills in the interdisciplinary field of Bioinformatic Analysis?

Yes, the Bioinformatics Club at Brigham & Women’s Hospital meets weekly to provide training and hosts speakers to support advances in this exciting field. If you’d like to join the club, please visit the website here: http://bioinformatics.bwh.harvard.edu

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