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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.
Consultation Services
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
The Core offers Agilent Fragment Analyzer 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 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 Fragment Analyzer and MiniSeq quality control, normalization, pooling, and sample submission to UCSF sequencing cores. We offer several standard protocols for sample preparation.
RNA-Seq
Universal Plus mRNA [Tecan]: this method includes a polyA selection step that generates strand-specific libraries covering protein coding RNAs (mRNAs) and other long polyadenylated transcripts. This method requires 10ng-1µg total RNA starting material.
Universal Plus mRNA [Tecan] + FastSelect ribo/globin Depletion [QIAgen]: these methods generate strand-specific libraries covering both polyadenylated and non-polyadenylated long transcripts. Requires 50ng-1µg 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.
SMART-Seq v4 [Takara Bio] + Nextera Flex [illumina]: these methods are appropriate when less RNA is available for analysis and can start with as little as 10pg total RNA.
ChIP-Seq
NexteraXT [illumina]: this method can start with as little as 1ng of chromatin immuno-precipitated DNA (supplied by the user).
Small RNA-Seq
Modified TruSeq Small RNA [illumina]: 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 10ng of total RNA.
Bioinformatic Analysis
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 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
Other bioinformatic analysis services are available, including single cell RNA-seq, ChIP-seq, WGS and more.
All analyses are available for download from UCSF's secure Box site. We schedule a meeting at the end of each study to discuss the data analysis in detail.