Our core offers support for all phases of functional genomics studies, including experimental design, sample preparation, quality control, and data analysis. We work through a recharge mechanism: investigators provide samples and we handle sample processing, quality control, and standard data analysis.
The core offers support for grant preparation, as well as consultation services for study design, sample preparation, and analytical approaches for data analysis.
Nucleic Acids Analysis
We offer Agilent Bioanalyzer quality analysis services for RNA and DNA samples that will be used for sequencing studies performed by the core. The user provides samples at the required concentration of 2ng/µl and the core facility staff will run the samples on a bioanalyzer chip and email the user our quality assessment and the data in a PDF file format.
Next Generation Sequencing Sample Preparation
The core prepares libraries that are suitable for sequencing on Illumina sequencers. Library preparation includes Bioanalyzer and MiSeq quality control, normalization, pooling, and sample submission to UCSF sequencing cores. We offer several standard protocols for sample preparation.
Illumina TruSeq Stranded mRNA: this method generates strand-specific libraries covering protein coding RNAs (mRNAs) and other long polyadenylated transcripts. This method requires 100 ng-1 µg total RNA starting material and a polyA selection step is included in the method.
Illumina TruSeq Stranded Total RNA with Ribozero Gold: this method generates strand-specific libraries covering both polyadenylated and non-polyadenylated long transcripts. Requires 100 ng-1 µg total RNA and a ribosomal depletion step is used to remove rRNAs, which would otherwise dominate the libraries. We also offer a version of this method that includes globin depletion and is useful for blood samples.
NuGen Ovation/NexteraXT: these methods are appropriate when less RNA is available for analysis, and can be used with as little as 500 pg total RNA.
NexteraXT: this method can start with as little as 1 ng of chromatin immuno-precipitated DNA (supplied by the user).
Modified Illumina TruSeq Small RNA: this method is designed to sequence miRNAs but also captures some other small RNAs and fragments of larger RNAs. We use random-end adapters to minimize ligation bias and optimized the method to work with as little as 10 ng of total RNA.
Our standard services include genomic alignments, data quality control and normalization, and identification of differentially expressed genes.
For RNA-Seq analysis, we offer:
- FASTQC quality control of raw sequencing reads
- Alignment to standard reference genome using rna-STAR
- Gene-level abundance measurements
- Sample-level unsupervised hierarchical clustering and principle component analysis
- Differential expression testing for pairwise, group comparisons as well as more complex models (multifactorial, time-series, etc) using DESeq2
- Integrative Genome Browser based visualization
All analyses are available for download from a secure FTP server. We schedule a meeting at the end of each study to discuss the data analysis in detail.
To initiate a project, please fill out the project description form (link to download form) and e-mail it to Andrea Barczak. When we receive the completed form, we will contact you to schedule an initial project meeting. During the meeting we will discuss experimental design, protocols, analysis needs, timeline, and cost.
We strongly recommend that you schedule a project meeting with us before you start your experiment so that you are aware of design issues that are likely to be important for the success of your project.
The core’s recharge system is used to cover labor and supply costs for most projects. Other arrangements, such as subcontracts, are available for larger or more complex projects.
Standard pricing is available for certain commonly used protocols. For other work, the core can give you a price estimate prior to starting on your project. Pricing is adjusted periodically to reflect changes in our costs. Users outside of the University of California system will be assessed a 26% surcharge.
RNA quality control
Core users prepare RNA samples and submit these to the core. When sufficient RNA is available for analysis (≥ 10 ng), samples should be analyzed by spectrophotometry (Nanodrop) and by electrophoresis (Agilent Bioanalyzer) prior to preparing sequencing libraries. Agilent Bioanalyzer analysis is offered as a service to users at a cost of $11.27 per sample.
RNA-seq library preparation
The core prepares libraries that are suitable for sequencing on Illumina sequencers. Library preparation includes quality control, normalization, pooling, and sample submission to UCSF sequencing cores. Sequencing costs are not included (see below). For projects with fewer than 4 or more than 48 samples, please contact us to discuss pricing.
|Library type||Method||Cost per sample|
|4-24 samples||25-48 samples|
|PolyA RNA||TruSeq stranded mRNA (>100 ng total RNA)||$393||$373|
|rRNA- and/or globin-depleted RNA||TruSeq stranded Ribo-Zero gold or globin (>100 ng total RNA)||$453||$430|
|Low input total RNA||NuGen Ovation v2 (>50 pg total RNA)||$478||$454|
|Small RNA (including miRNA)||TruSeq small RNA (>10 ng total RNA)||$376||$357|
|ChIP-Seq library prep||NexteraXT (>1 ng DNA)||$223||$211|
We submit libraries prepared in our core to the UCSF Center for Advanced Technology and the UCSF Institute for Human Genetics Genomics Core. Both Cores have Illumina HiSeq instruments that can run in high output and rapid run modes. Prices are determined by these cores and change periodically to reflect changes in reagent and other costs. We typically aim for 40-50 million reads per sample for standard RNA-Seq projects. Sequencing costs are approximately $150/sample for single-end 50bp sequencing and $400/sample for paired-end 100bp sequencing.
Analysis pricing is charged on a per project basis and is determined by the complexity of the experimental design. Typical costs range from $900-$2500.