Q: Where is the core located?
A: Rubin 670 in the Norris Cotton Cancer Center, Lebanon, NH
Q: How do I initiate a project?
A: We strongly encourage all users to speak with a scientist in the Shared Resource before beginning a new project. Contact information for scientists with the Genomics, CRISPR and Dartmouse services can be found on the 'Services' page. Through this initial consultation, we can help you to select a technology and workflow best suited to answering your research questions and provide pricing information to ensure the service fits within your budget.
Q: How to I place an order?
A: All orders are placed through the RaDar system. Please complete any required order forms for the service and provide an active account number. It is also recommended to email a member of the Shared Resource to confirm receipt of the order.
Q: What types of samples are suitable for RNA extraction?
A: RNA can be isolated from 1,000 to >1 million cells and from most tissue/fluid types. FFPE samples can be processed as well but reduced yield and quality are expected.
Q: How should samples be submitted for RNA extraction?
A: Small numbers of cells (<100,000) should be FACS sorted, or otherwise deposited into 200ul Qiagen RLT buffer and stored at -80C until submitted. Users should coordinate with the GSR to ensure samples can be processed in a timely fashion to prevent RNA degradation and reduced yields. Larger numbers of cells can be pelleted and resuspended in 200ul RLT buffer and stored at -80C until submission. Tissue samples can be stored in RNAlater at -80C until submission.
Q: How can I compare the different types of RNA-seq offered and the associated costs?
A: We have created a tool to help you understand the types of RNA-seq and their costs. The tool can be downloaded HERE. You can (and should!) also schedule a consultation with a scientist in the Shared Resource to go over the specific needs of your project.
Q: How many replicates should I perform per sample?
A: We strongly recommend at least 4 biological replicates per experimental condition for all experiments.
Q: I have FFPE samples I would like to use for sequencing or microarray analysis. Will these work?
A: FFPE samples can vary greatly in quality and likewise in their performance in NGS and microarray workflows. Quality control becomes particularly important for these samples to prevent wasted time and resources and to ensure the success of the experiment. RNA samples are run on the Fragment Analyzer to determine the size distribution of RNA fragments and a DV200 score is calculated to identify potentially problematic samples. DNA samples are subjected to a qPCR QC assay to assess functional performance of the material in comparison to a validated control sample. These precautions greatly increase the probability of generating high quality data from an otherwise challenging sample type.
Q: I am interested in a custom assay such as amplicon sequencing or a custom microarray. Can the GSR handle this?
A: Yes! We are happy to work with you and the vendor to design sequencing or microarray panels that will suit your needs and perform the workflow, including the necessary QC steps. The user is responsible for ordering the custom reagents while the GSR charges for the common reagents and labor required to prepare the samples.
Q: Will you sequence libraries I have prepared myself?
A: Yes! You can provide us with pre-pooled and normalized libraries that are ready for sequencing, or we can perform library QC, normalization and pooling for you. For pre-pooled libraries we are not responsible for issues with sample balancing or data yield. You should also provide an Illumina sample sheet, or a list of the index sequences used for each sample so we can deliver demultiplexed fastq files.
Q: How are data generated in the GSR shared with users?
A: Data generated in the GSR is stored on the Dartmouth DartFS file storage system, which can be accessed through the Discover/Andes/Polaris computing environments, or mounted as a network drive on your computer. Each lab is provided a folder where there data is stored and whose access is restricted to NetIDs approved by the PI. In the event that the lab does not use the DartFS system, or data needs to be shared with a collaborator outside Dartmouth, a web server is provided with links to download the data. Please let us know if you require this service.
Q: How long is data generated in the GSR retained?
A: Data generated in the GSR is stored as demultiplexed fastq files for 1 year on high-speed disk space in DartFS in a read-only format to prevent alteration of the raw files. These files can be copied to the users' own disk space for manipulation, or can directly serve as input into downstream analysis pipelines to prevent data redundancy. After 1 year, fastq files are removed and the raw .bcl sequencing files are retained in archival storage for 4 additional years. If needed, the GSR can provide access to the archived files should the original data be lost or otherwise corrupted. Beyond 5 years, data storage becomes the responsibility of the user.