The facility provides library preparation and then high throughput (next-gen) DNA sequencing using an Illumina NextSeq 500. Automated samples preparation is carried out with an Eppendorf Epimotion. The Core supports transcriptome and exome analysis, ChIP-seq, and a variety of other approaches. Single cell sequencing analysis is now offered using the Illumina/Bio-Rad ddSEQ system and the 10X Chromium systems for library preparation. RNA quality analysis is performed utilizing Bioanalyzer, NanoDrop, and Qubit instruments. Spatial Transcriptomics is supported by a Nanostring GeoMx, with the upcoming introduction (mid-2022) of true single transcriptomics with the Nanostring CosMX instrument.
Next gen sequencing
The facility provides high throughput sequencing on the Illumina NextSeq 500 sequencer, which is capable of producing up to 400 million reads per run, with run times ranging from 19-29 hours. The NextSeq 500 is very scalable allowing it to support several applications including, sequencing of small genomes, targeted sequencing, whole transcriptome, ChIP-seq, and whole exome sequencing. The core offers full service library preparation, and sample QC. Included in the cost of sequencing is basic bioinformatic analysis through Illumina BaseSpace (SNP/InDel calling, transcript abundance). The core also provides advice on experimental design, and guidance on the capabilities of next-generation sequencing. Advanced bioinformatic and systems biology analysis is available through the Bioinformatics Core in La Jolla.
Single cell RNA-SEQ and ATAC-SEQ
The core is equipped with two single cell systems, the 10x Genomics Chromium Controller, and BioRad ddSeq single cell isolator. Core staff can assist with processing single cell suspensions, library prep, sequencing, and basic informatic analysis.
NanoString GeoMX Digital Spatial Profiler
The GeoMx is used to spatially resolves RNA or proteins, up to the whole transcriptome or 96 proteins within multiple regions of interest on FFPE or FF tissue sections. These ROI can be defined by first staining the tissue sections with antibodies that identify various tissue compartments or cell types of interest.
A NanoString GeoMX Digital Spatial Profiler can spatially resolve RNA or proteins at high multiplex within multiple specific regions of interest. True single transriptomics will be upcoming with a NanoString GeoMx instrument.
Equipment & Resources
- Illumina NextSeq 500
The NextSeq 500 Sequencing System from Illumina provides the widest range of sequencing applications. The flexible, scalable system offers tunable read lengths and output options to support research needs. It can sequence 20 RNA-seq, or ChIP-seq libraries per day and supports Whole-Genome Sequencing and Exome Sequencing.
- 10X Genomics Chromium Controller Single Cell System
The Chromium is used to prepare single cell RNA-seq and ATAC-seq libraries from single cell suspensions provided by users. The Chromium is capable of processing from a few hundred to 5,000 cells per sample. Core staff can train users on the system, or assist with sample preparation.
- Bio-Rad ddSeq Single-Cell Isolator
Like the Chromium, the ddSeq is a single cell RNA-seq and ATAC-seq library preparation system. The ddSeq is well suited for pilot studies with less than 1000 cells targeted per sample.
- Eppendorf epMotion 7075 Liquid handling system
The epMotion 7075 is used for high throughput construction of RNA and DNA libraries for sequencing on the NextSeq 500.
- Agilent TapeStation 4200
The Agilent 4200 TapeStation system provides sizing, quantitation and quality control of DNA and RNA. It is especially useful for assessing the integrity of submitted RNA samples.
- Qubit Fluorometer
The Qubit Fluorometer gives an extremely accurate measurement of DNA, RNA or protein.
- Nanostring nCounter-Amplification-free RNA Analysis
The nCounter is an excellent instrument for analyzing the expression of a substantial number (e.g., 100) of genes and micro RNAs in a large number of samples, such as validation of putative biomarkers in clinical samples, including FFPE samples.
- Roche LC480 real time QPCR instrument (interchangeable 96 or 384 well)
- Roche LC96 real time QPCR instrument
- Stratagene Mx3000p real-time QPCR instrument (96 well)
- Nanodrop Spectrophotometer
- Shared Instruments:
- Roche LC480 (2) QPCR (interchangeable 96 or 384 wells)
- LC96 (4) QPCR
- ABI 7900HT real time QPCR (interchangeable 96 or 384 wells)
- Nanodrop spectrophotometers (3)
For a complete list of services, please call (858) 646-100 ext. 3714 or email us.