1. Single Cell Analysis
1.1 Single Cell (sc) Genomics Assays:
scRNA-seq libraries are generated from 100 to 10,000 individual cells captured in an oil emulsion, cDNA is generated in the individual cell-gel bead emulsion micro-reactors while adding barcodes at the cellular and molecular level. The barcoded cDNA from the individual cells is combined for the remaining library process. The unique molecular barcodes (UMIs) ensure that amplification artifacts do not skew the analysis. The libraries are sequenced on an Illumina sequencer.
· sc-Immune Repertoire Analysis simultaneously analyzes V(D)J repertoire and gene expression in 100 to 10,000 individual cells. A 5’ gene expression library is produced from captured cDNA similarly to the scRNA-seq described above. 5% of the captured cDNA is used in a targeted enrichment amplification to produce a library, which enables sequencing of full-length V(D)J segments including alpha and beta T-cell receptor or heavy and light chain immunoglobulin sequences from B cells.
· REAP-seq or RNA Expression and Protein Sequencing assay uses barcoded antibodies to label cells prior to scRNA-seq allowing simultaneous mRNA and Protein measurements.
· Perturb-seq - an scRNA-seq screen of cells that have been transfected with a CRISPR library of barcoded guide RNAs.
· scCNV-seq captures individual cells in gel beads for cell lysis, trapping DNA from each single cell DNA in the gel beads. It enables detection of CNVs at 2Mb resolution, down to 100s of Kb in cell clusters and allows analysis of mosaicism, cancer genomics and genetic disorders at a cellular level.
1.2 Single Cell Western analysis – A single cell suspension is loaded onto a microscope slide that is coated with a gel matrix layer. Wells in the layer are sized to capture single cells, then the cells are lysed and the protein content of the cells are electrophoresed to produce a chip that contains up to 1000 single cell Westerns. The chip can be probed multiple times with standard Western antibodies using spectral and size-based multiplexing, stripping and re-probing. The chips are scanned with an Agilent 2505 microarray scanner. The core encourages users to consider making a western chip with the same cell samples that are used for scRNA-seq. This allows users to validate scRNA-seq data with protein expression in single cells. The chips may be archived for up to 9 months allowing ample time for scRNA-seq analysis. Single-Cell Westerns can also be used to study post-translational modifications such as phosphorylation that are not revealed by scRNA-Seq analysis. In addition, this method can detect proteins that lack good flow cytometry antibodies, protein isoform heterogeneity and handle samples that are too small for FACS or bulk Westerns.
1.3 Targeted Single Cell DNA-Seq - The Fluidigm Biomark platform is used to generate targeted DNA-seq libraries from single cells. Users capture up to 48 single cells in the wells of a microplate by flow cytometry. The DNA of the single cells undergoes Whole Genome Amplification in those wells and are then loaded into a Fluidigm Access Array chip. Up to 48 PCR primer pairs or 48 pools of up to 10 pairs of primers are loaded into the chip and combined with each sample in picoliter size reaction chambers. The amplified targets are harvested from the chip and barcoded with adaptor primers for sequencing in an Illumina MiSeq. Up to 12Kb (non-contiguous) may be targeted without multiplexing primers, depending on target size, up to 384 samples, from multiple Fluidigm chips, can be combined into one MiSeq lane.
2. MPS (Next Gen Sequencing) – Please see the Epigenomics Shared Resource website (link is in sidebar on the right) for information on MPS assays including RNA-seq, ChIP-seq, ATAC-seq, MethylC-seq, HELP-tagging and MAD-seq.
3. Gene Expression Microarrays
Affymetrix microarrays are used for gene expression studies when a fast turnaround and high sensitivity are required. Current arrays have higher sensitivity for low abundance transcripts compared to low depth RNA-seq. The increased content of these arrays can quantify or detect alternative splicing, directional gene expression and non-coding RNA. Multiple options for RNA amplification are offered to accommodate projects with very limited sample sources, from 100 picograms to 100 nanograms of total RNA. A qPCR assay with a standard curve is used to quantify low concentration RNA samples. This assay is also used to qualify FFPE RNA; the qPCR assay is more predictive than an Agilent RNA Integrity Number (RIN.)
· MicroRNA arrays at a very low cost allow researchers to focus on small non-coding RNA or to pair them with whole transcriptome expression arrays.
4. Genotyping and Copy Number Variation (CNV) Microarrays -Thousands of DNA samples have been processed at the core using the Affymetrix SNP 6.0 chip, which has 1.8 million DNA variants divided roughly into SNP and CNV probe-sets. These are used in genome-wide association studies (GWAS) and DNA copy number analysis.
5. Single Nucleotide Polymorphism (SNP) and Mutation Analysis
The facility uses a MALDI-TOF mass spectrometer (Agena Bioscience, San Diego) for custom SNP genotyping to validate variants found by MPS or to replicate top SNPs in GWAS, or to screen targeted panels in large numbers of samples. The iPlex application uses single base extension to analyze up to 36 DNA variations in a multiplex reaction. This service includes assay design, oligonucleotide prep followed by automated PCR setup so that users only need to submit genomic DNA and a SNP list. 100s to 1000s of samples are easily analyzed with this method.
· Rare mutations in tumors and plasma as low as 0.1% frequency may be detected with an allele specific oligo variation of the iPlex application.
· Taqman SNP assays are used to for projects with a small number of SNPs and for SNPs that cannot be assayed by the single base extension method.
6. Cytosine Methylation Analysis - Cytosine methylation analysis (Epityper) is performed on the MALDI-TOF instrument. DNA fragments of up to 600 base pairs are analyzed after bisulfite PCR from genomic DNA. The method can detect the percent of methylation at most CpG sites in the fragments. Epityper is also used to validate epigenomic MPS studies.
7. Sanger Sequencing - for traditional DNA sequencing of premixed template and primer using standard primers provided at no cost or custom primers supplied by the investigator. Automated reaction cleanup with CleanSeq (Beckman Genomics) allows fast turnaround. For amplicon sequencing, PCR purification with Ampure (Beckman Genomics) is performed on the same automation platform. Typical read lengths approach 1000 bases. Quality values are assigned to base-calls and data is returned via a password-protected ftp server within 24 hours for the majority of samples.
8. Cell Line Authentication (CLA) - CLA of human cell lines is recommended annually, when cell lines are transferred between labs and when unverified stocks are thawed. Users submit purified DNA or cells spotted on an FTA card (Whatman). The facility amplifies the DNA with fluorescently labeled Short Tandem Repeat (STR) markers; electrophoresis and detection are done on the 3730 DNA Analyzer. Positive and negative controls and an allelic ladder are included in each batch. The allele calls are reviewed by experienced personnel and are compared to two cell line databases, one maintained by The American Type Culture Collection (ATCC), the other by the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). The user receives a report with the matches to the databases, the alleles for each marker in the test and the chromatograms(the raw data).
9. Microsatellite Instability Analysis (MSI) - The facility service includes electrophoresis and detection of fluorescently labeled microsatellite markers provided by the users. The data is analyzed with GeneMarker software (SoftGenetics, State College, PA) to score the difference between tumor samples and normal controls.
10. RNA/DNA Quality Analysis – A Fragment Analyzer or Bioanalyzer (Agilent, Santa Clara, CA) is used to assay the quality of RNA intended for MPS or microarray assays. These are the primary tools used to check libraries prior to loading on Illumina sequencers. Analysis is usually performed the day of submission and others are completed within 24 hours.
11. qPCR - The facility offers access to our qPCR instruments and instruction on their use. Researchers may reserve the machines in iLab to run their plates. Investigators may also use the facility to perform all steps of the qPCR process, from assay design to sample processing. Consultation and troubleshooting are also available.
12. Liquid Handling Automation - The facility can automate liquid handling to facilitate your high throughput experiments. Examples of processes that can be automated include PCR setup, DNA/RNA normalization, sample labware reformatting (e.g. from tubes to 96 or 384-well plates.)
13. DATA ANALYSIS – Data is made available to researchers and their PIs through Dropbox. The data is screened with appropriate quality control measures. Assistance with analyzing data, instructions as to how to use the tools and more extensive data analysis on a fee-for-service basis are provided by the Computational Genomics Core.
