FAQ

What are the advantages of working with the core instead of doing the experiment in my own lab?

Core staff have extensive experience with all phases of functional genomics analyses, including experimental design, laboratory protocols, and data analysis. The core is equipped with specialized equipment, including library preparation automation, and we purchase reagents in quantity and pass the savings on to our users.

Can you help me prepare a grant proposal involving experiments to be performed in your core?

Yes, we are happy to help you prepare your proposal. Please contact us if you require a support letter or specific experimental details. You may find the following descriptions of the core facility useful for preparing your proposal:

Facilities:

The UCSF Functional Genomics Core Facility is directed by David Erle, MD, Professor of Medicine, and located adjacent to his laboratory. The facility, Dr. Erle’s laboratory, and adjacent shared support rooms, occupy a total of ~3000 ft^2 on UCSF’s Mission Bay Campus. The core is staffed by a full-time manager with >15 years of high throughput genomics experience and by staff members with expertise in sample preparation, quality control, and data analysis.

Office: Dr. Erle’s office is located on the same floor as the Functional Genomics Core and his laboratory.

Computing: The Functional Genomics Core Facility currently operates two in-house workstations containing dual 2.93 GHz 12 core/24 thread Intel Xeon X5 processors and 96-128 GB of 1066 MHz RAM.  These workstations contain internal hard drives totaling 6 TB of storage space and are connected to a 32TB Pegasus 2 Raid 5 enclosure.

Equipment:

Equipment in the UCSF Genomics Core Facility, Dr. Erle’s laboratory, and adjacent shared equipment rooms includes a state of the art Beckman-Coulter SPRIworks High Throughput automation system (based on the Biomek FXP system, and configured for Next Generation Sequencing library preparation, nucleic acid isolation, and automated PCR in a 96-well format), an Agilent Bioanalyzer, an Advanced Analytical Fragment Analyzer, a multichannel Nanodrop spectrophotometer, a PippinPrep size selection instrument, 5 cell culture hoods, 10 cell culture incubators, inverted light and fluorescence microscopes, multiple PCR machines (including a 384-well plate instrument for TaqMan and SyBr green qPCR), -80 and -20 °C freezers, liquid nitrogen tanks, refrigerators, a cold room, a 37 °C warm room with shakers for bacterial culture, and centrifuges. The core has full access to multiple Illumina sequencers including the NovaSeq, HiSeq 2500 and 4000 MiniSeq, and MiSeq systems at both the Mission Bay and Parnassus campuses. We also have full access to a Fluidigm Biomark microfluidics-based nanoliter multiplex qPCR system, and multiple flow cytometers operated by the UCSF Laboratory for Cell Analysis.

How long will it take to get my results?

Studies that utilize standard protocols take about 8-10 weeks to complete from submission of RNA to data analysis. We will provide an estimated timeline for all projects when we meet with you before starting your project.

How much will my project cost?

Pricing information is provided here .

Which parts of the project are my responsibility and which parts are the core’s responsibility?

Users prepare high quality RNA and submit the samples to the core in a 96 well plate format at a specified concentration and volume  (this is determined by the core based on your RNA yields and quality metrics). The core will prepare, quality control, normalize and pool the sequencing libraries, submit libraries for sequencing, and analyze the sequencing data.

Do you offer “a la carte” services? For example, can you sequence libraries made outside your core or analyze genomics data generated outside of your core?

The core generally prepares sequencing libraries and performs data analysis for every project. For RNA samples requiring Bioanalyzer analysis, users may perform this analysis themselves or submit to the core for Bioanalyzer analysis. Data analysis may be performed outside the core, although in these cases initial data analysis will generally be performed by core staff as part of the quality control process.  

How do I acknowledge the core in my publications?

Thanks for asking! We like to consider our projects as collaborative ventures between core users and core staff. Please consider including core staff members as co-authors if they made suitable contributions to your work. Please mention the UCSF Functional Genomics Core Facility and consider naming core staff members who made contributions but are not co-authors in the acknowledgements section. We are happy to help prepare manuscripts and appreciate being notified when your manuscripts are accepted for publication so that we can include your work on our Publications page .

Experimental design

How many replicate samples should be in each experimental group?

This depends. Generally technical replicates (e.g., multiple sequencing libraries prepared from the same sample) are not helpful, but biological “replicates” (multiple samples from different individuals or different cultures) are important. In most cases we recommend a minimum of 3-4 samples per group, but more samples are advisable if the population is heterogeneous or the expected differences are small.

What kinds of control samples are appropriate?

In general, the same issues apply to selecting controls for genomics studies as apply for other kinds of experiments. However, it is important to remember that genome-wide assays are very good at detecting changes due to “nuisance” variables that are not related to the experimental variables that you wish to study.

I cannot generate all my samples at the same time. How should I process  them?

If you can not process all of your samples at the same time, we recommend that you evenly distribute samples from different groups across batches when generating samples and extracting RNA. For example, if you have 16 samples (4 groups of samples, each with 4 biological replicates) and can only process 4 samples at the same time, you should process 1 sample from each of the 4 groups in each batch.

How do I determine what kind of sequencing library preparation is appropriate for my project?

Information about the standard library preparation methods is provided here ( link to RNA-seq library preparation

On services/our-services page). We will discuss this with you at the project meeting to ensure that we use the most appropriate protocol for your experiment.

 

Preparing samples for submission

What RNA isolation methods are suitable for RNA-Seq?

Most RNA extraction methods can be used for RNA-Seq, although some methods may be more suitable for certain types of samples or library preparations.

Here are some factors that you should consider before deciding on the extraction method for your samples:

1)    The type of information are you primarily interested in assaying (small RNA, mRNA, long-noncoding RNA).

2)   Type of starting material (cells/tissue/etc).

3)  Amount of starting material ( i.e., hundreds of cells, mg amounts of tissue)

We recommend consulting with the Core staff before you set up your experiment.


 

Quantity and Purity

What kind of quality control should be used for RNA samples?

If total RNA is extracted from samples and is more than 2ng/ul, the first Quality Control assay to complete is the NanoDrop spectrophotometer to measure the concentration (ng/ul), the protein contamination (260/280) and organic compound contamination (260/230).

Ideally the two ratios will be >1.8 for all samples, however we can proceed with samples that have less than ideal ratios, please consult with the core if your ratios are lower the 1.8.

Quibit fluorometric quantitation can be used to measure more dilute samples.

When RNA is too dilute to measure accurately, we recommend normalizing the amount of input across your samples (if possible) and using and the entire eluate for library preparation (this may require concentrating your sample, please consult with the core before proceeding).

 

Quality

RNA integrity is assessed using the  Agilent Bioanalyzer RNA Pico Chip or the Advanced Analytical Fragment Analyzer.  Collaborators can complete this QC step if they have access to either of these instruments at their facility.  All UCSF collaborators have access to this equipment at the Center for Advanced Technology (http://cat.ucsf.edu). This service is also offered by the core to our collaborators, it will add a one week to the turnaround time.

RNA is measured by nanodrop, diluted to specified concentration and submitted for QC..

The results are saved in a pdf format and emailed to you with our assessment of the RNA quality and our recommendations.

How do I deliver samples to the core?

Please contact the core to set up a time for sample drop off.


 

Data analysis

What kinds of results do I get at the end of my project?

Most core users will receive an Excel spreadsheet containing all Ensembl annotated genes, fold changes between specified groups, statistical cut-offs for assessing differential expression and normalized read counts along with relevant figures such as heatmaps, pairs plots, etc.  In addition, we provide various QC metrics for assessing quality of both the sequencing run and samples themselves.  All raw and processed data will also be provided.

Can the core perform more advanced, customized analyses for my project?

Because of the volume of projects processed by the core, we do not generally accept customized analysis requests.  If, however, the analysis in question is limited in scope, feel free to email us with specific questions.

How do I submit my samples to public databases?

Most journals now require data to be submitted to either Gene Expression Omnibus or Short Read Archive as a condition for publication.  Both archives offer detailed submission instructions.  If you need assistance with part of the process, please contact us.