Bio-Rad ddSeq


Overview

For labs working with infectious agents that are restricted to BSL2 labs, the UMGC has placed a Bio-Rad ddSeq single-cell isolator in a BSL2 lab for DIY access, giving researchers who work with HIV, influenza, mycobacterium, etc. access to a droplet partitioning technology. When combined with the Illumina SureCell WTA 3’ kit, the ddSeq is a scalable and high-throughput sequencing solution that allows transcriptome analysis on individual cells to give a high-resolution, unbiased profile between cell populations. It uses an end-to-end workflow that starts with capturing single cells in nanoliter-sized droplets in disposable cartridges for up to four samples and ends with BaseSpace analysis with the SureCell RNA Single-Cell app, which calculates and reports single-cell metrics.

ddSeq workflow

Training


The UMGC provides training on an as-needed-basis in the BSL2 lab located in the Microbiology Research Facility building near TCF Stadium. A staff scientist, who specializes in single-cell technologies, will guide new users through Day 1 of the SureCell WTA 3’ protocol, including isolating and barcoding single cells through 2nd strand synthesis. Training will be hands-on using a cell suspension, a SureCell WTA 3’ kit, and consumables provided by the researcher and up to two scientists from one lab can attend the ~4-hour training at $162.58/lab. For researchers needing training on the complete library prep protocol (Day 1 and 2), please inquire.

Reserving the ddSeq


After the training run, researchers should be able to independently use the ddSeq isolator for isolating and barcoding single cells without further support from the UMGC staff.

DD seq

Jerry Daniel at single-cell@umn.edu will assist in coordinating a time with the researcher and the BSL2 lab. Please be sure to have a confirmed time at the ddSeq workstation before preparing your cell suspension – it can take up to a week to coordinate the BSL2 workstation.

Cell Preparation


The average cell output is 300 single cells/chamber that start with an input suspension of 2,500 cells/ul in 1X PBS + 0.1 % BSA solution, resulting in a 3-4% capture efficiency. It is critical for the ddSeq isolator to have a cell concentration between 2,250-2,750 cells/ul for optimal performance.

In addition, researchers should prepare a high-quality, clump-free cell population that has >95% viability and contains intact cells – broken or dead cells will release nucleic acid in the cell suspension and increase background signal. Cells must also be dissociated and free from aggregates to prevent doublets or multiplets in the droplets, and to prevent this, it is recommended to run the cells through a 35 µm cell strainer.

The ddSeq workflow has been tested with mouse and human commercially available cell lines and primary keratinocytes.

Purchasing reagents


The SureCell WTA 3’ kit can be purchased in 8-sample or 24-sample configurations through Adam Hauge at Illumina (ahauge@illumina.com) and researchers will receive the UMN discount. Illumina guarantees the shelf life to be at least three months from the date of receipt. Additional consumables are the responsibility of the user.

Library QC


Final libraries are submitted to the UMGC for QC analysis, including size determination on the BioAnalyzer High Sensitivity chip, and quantification by a fluorescent method as well as quantitative PCR. Libraries with a broad size distribution between 300-1,000 bp, with no fragments in the 100-300 bp range, will be recommended for sequencing. Based on the QC results, passing libraries will be diluted and pooled by the UMGC for sequencing.

Questions?


Please see the Guidelines tab for the Illumina SureCell WTA 3’ Reference Guide. For additional questions on using the ddSeq in the BSL2 lab, contact Jerry Daniel at single-cell@umn.edu.

 

Pricing

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Standard

Service Scale Unit UMN External
Required training. - 1/2 library prep protocol.
A UMGC scientist will guide new users through isolating and barcoding single cells through 2nd strand synthesis. Up to two scientists from one lab can attend the training. 1 session $162.58 $186.92
Required training - full library prep protocol.
Training on the complete SureCell WTA 3’ library prep protocol starting with single-cell isolation through library QC. 1 session inquire N/A
Client operated ddSeq run.
Client independently uses the ddSeq workstation in the MRF BSL2 lab to fully execute their own experiment including barcoding single cells and library prep. 1 day $317.72 $365.27
Guidelines

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Getting Started

How to sign-up


The UMGC provides training as needed in the BSL2 lab located in the Microbiology Research Facility building near TCF Stadium. Training is scheduled through Jerry Daniel at single-cell@umn.edu and users supply cells, kits, and consumables.

Once ddSeq training is complete, users are able to reserve the ddSeq in one-day increments. Jerry Daniel at single-cell@umn.eduwill assist in coordinating a time with the researcher and the BSL2 lab. Please be sure to have a confirmed time at the ddSeq workstation before preparing your cell suspension – it can take up to a week to coordinate the BSL2 workstation. Clients should email the below reservation form to single-cell@umn.edu to provide an EFS number for billing and indicate the confirmed day and start time for the ddSeq workstation.

Forms


Client Instrument Reservation Form

 

Client Consumable List

 

SureCell WTA 3’ Reference Guide

 

Location for training and the ddSeq


BSL 2 Lab
Microbiology Research Facility (MRF) Building
Lead: Jerry Daniel
Phone: 612-625-6175
Operating Hours: 9:00 AM – 5:00 PM Monday – Friday

Deliverables

Data Release


There are three options for transferring data from the UMGC to clients: delivery to the Minnesota Supercomputing Institute’s (MSI) high-performance file system, download from a secure website, or shipment on an external hard drive. Please indicate your data delivery preference when placing a order for sequencing.

MSI storage

Internal clients and external clients that opt-in to MSI storage have their data released to the client’s MSI group directory on the MSI high-performance filesystem (/home/GROUP/data_release/umgc), from where it can be analyzed using MSI’s high-performance computers or Galaxy system. The data is stored for five years in uncompressed format with read-only permissions to avoid accidental modification, and secure off-site tape backups of the data are maintained for disaster recovery. MSI will charge the client a storage fee for this service.

Web download

Internal clients that opt-out of MSI storage and external clients can download their data from a secure website using either a web browser or a command-line download tool, complete instructions are provided in an email from the UMGC. The client’s data is available for download for 30 days, after which the data will be removed from the data download website and the client takes responsibility for storing the data.

Hard drive

External clients may have data shipped on a hard drive purchased by the UMGC and invoiced to the client.

Data Recovery


MSI storage

MSI guarantees storage of client data for five years, after which the data can be transferred to tape for long-term storage, contact MSI (help@msi.umn.edu) for more information.

Web download and hard drive

Data is typically recoverable for up to 5 years. If the external researcher should ever need the FASTQ files re-posted to the data download website or shipped on a hard drive within 5 years from data release, email next-gen@umn.edu to initiate data recovery. The client will be charged for the cost of recovering the data from tape archive. The UMGC does not provide any guarantee that data can be successfully recovered from tape archive.