The Genomics Shared Resource serves as a hub for consultation and scientific interaction relating to nucleic acid-based methods. It provides expertise and support to insure the best possible outcomes for genomic related projects. The Facility supports several state-of-the-art platforms for a wide variety of nucleic acid-based studies, including massively parallel sequencing as well as routine capillary sequencing. The Facility also supports transcriptomic projects including RNA-Seq, ChIP-Seq, methylation (MeDIP), small RNA-Seq and targeted sequencing. In addition, we support gene expression studies using Quant-Seq (3’ RNA-seq) and low input sequencing for most applications. Single Cell-Seq is supported by the Takara iCell 8 MSND system. Additional platforms for targeted gene expression studies include the nanoString platform supporting all custom or commercially available applications. We also provide SNP genotyping using TaqMan assays, microsatellite analyses and C. bovis assays. RNA/DNA isolations from various types of samples, including FFPE are also provided.
In addition to consultation and collaboration with Wistar Cancer Center members, the Facility provides services to the greater scientific community.
The establishment of this facility was supported in part by an NCI Cancer Center Support Grant and equipment grants from the Commonwealth of Pennsylvania, The Pew Charitable Trusts and the National Cancer Institute."
"The ABI 3130xl is a 16 capillary machine that can sequence over 100 samples/day. The 3100xl can also be configured for fluorescent fragment detection and microsatellite analysis and is equipped with Gene Scanning Software."
"The Agilent 2100 Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells. Results are delivered within 30-40 minutes in automated, high quality digital data.
Miniaturization of analytical instrumentation has many advantages over conventional techniques. These advantages include improved data precision and reproducibility, short analysis times, minimal sample consumption, improved automation and integration of complex workflows. Launched in 1999, the Agilent 2100 Bioanalyzer was the first commercially available instrument to use microfluidics technology for the analysis of biological samples. Today, the Bioanalyzer offers a broad range of pre-validated analysis kits combined with an easy-to-use benchtop system."
"ImageQuant LAS 4000 is a digital imaging system for sensitive, quantitative imaging of gels, blots, and colonies. ImageQuant LAS 4010 Fully equipped imager that in addition performs ultraviolet (UV) and visible (RGB) fluorescence imaging."
The Illumina BeadStation 500GX has an autoloader, manual and DASL options. Infinium whole genome genotyping can be carried out on the BeadStation.
"Illumina’s Genome AnalyzerIIx is a proven platform for genetic analysis and functional genomics, and has transformed the way experiments are developed and executed. Massively parallel sequencing technology leverages clonal cluster formation and proprietary reversible terminator chemistry to dramatically improve the speed, and reduce the cost, of large-scale sequencing."
"The Genome AnalyzerIIx supports a wide range of applications, including whole-genome and candidate region resequencing, transcriptome analysis, small RNA discovery, methylation profiling, and genome-wide protein-nucleic acid interaction analysis."
"The Qubit® 2.0 Fluorometer is Life Technologies’ second generation fluorometer designed to work seamlessly with our highly specific and sensitive Qubit® DNA, RNA, and protein quantitation assays."
Available upon request
ABI OpenArray miRNA assay is available for human and mouse plates. OpenArray digital, pathway specific PCR and custom miRNA and gene expression assays are available upon request.
"The facility uses an ABI 3130xl capillary machine for Sanger-type sequencing with fluorescently-labeled dye-terminators, the ABI four-color detection, and sequence analysis system."
"Purified DNA is provided by the investigator with an order form specifying the quantity, form (double-stranded, single-stranded, or PCR product) and estimated size of the DNA. It is essential that the size and concentration of PCR products are determined accurately prior to sequencing. Lambda or Bac clones require submission of greater quantities of DNA."
"For each sequencing run, investigators must provide in numbered or labeled 1.5 ml Eppendorf tubes:
Double-stranded DNA: 1-1.5 µg of purified DNA at 0.2-0.25 µg/µl
Single-stranded DNA: 1 µg of purified DNA at 0.1-0.125 µg/µl
PCR products: at least 15ng of purified DNA per 100 bp of PCR product in 5ul water
Lambda phage or Bac clones: 10ug purified DNA at 2-2.5 µg/µl
A name designation of 8 or fewer characters and the primers to be used for each run must be clearly indicated on the submission form.
M13 forward, M13 reverse, T7, T3, and SP6 primers are provided by the Facility at no additional cost. Specific primers must be supplied by the investigator at 3.2 pmoles/µl.
Microsatellite Analysis: at least 450ng of DNA at 30ng/ul for 5 loci assay, and 10ng of DNA at 0.125ng/ul for 16 loci assay.
Depending on well availability, DNA samples delivered by 11:00 am will be available the next day at 3:00 pm."
"For clean double-stranded templates, extremely accurate sequence reads of >=600 bases are obtained routinely. Text files of sequence data are e-mailed. The original result folder from the ABI base caller is placed in a computer folder for electronic retrieval if needed.
Other modes of delivery can be arranged."
DNA isolation using Tri-reagent.
Other Services offered by the Genomics Facility include:
Protocols: RNA preparation, hybridization, RNA amplification
Troubleshooting: Send an email with your questions to firstname.lastname@example.org.
Training: By appointment only, hourly cost basis
Grant applications: Core descriptions and Facility generated array figures can be provided for grant applications with sufficient notice. Email Dr. Louise Showe with questions.
Illumina Next-Generation Sequencing (NextSeq 500 and Hi-Seq2000)
-- Gene Expression Analysis: Lexogen QuantSeq 3’ mRNA-Seq library prep kit is used to generate Illumina compatible libraries.
-- Transcriptome analysis applications: RNA-Seq, RNA-IPSeq, small RNA-Seq
-- Genome sequencing
-- MeDIP and hMeDIP
-- Exome and Amplicon sequencing
"Gene Expression Arrays (for human and mouse, including every step from RNA purification, amplification through hybridization and image quantitation)
• GoldenGate Genotyping Technology
• Infinium Whole-Genome Genotyping
• Gene Expression in FFPE samples
• Human promoter Infinium methylation studies from DNA purification and bisulfate conversion"
• DASL gene expression for degraded RNA (with or without RNA isolation)
• mouse low density (Ld) linkage panel
"Investigators are asked to choose the Illumina Beadchips that best suit their needs and provide information about the quantity and quality of the RNA being submitted. The average time required for hybridizations, starting with sample submission and ending with an image of the array, is 10 working days; however, this time will vary based on the numbers of samples and the quality and quantity of RNA or DNA."
"Nanodrop and Bioanalyzer analysis can be provided."
nanoString nCounter platform:
-- Multiplexed digital quantification of nucleic acids without sample amplification
-- Applications for gene expression, single cell, miRNA, IncRNA, CNV and CHIP/RIP projects
RNA amplification using Ambion or Epicentre kits. Amplification plus labeling is carried out with NuGEN kits.
RNA isolation using Tri-reagent, RNAqueous Micro kit, or Paxgene kit. RNA isolation ribbons using High Pure RNA Paraffin kit.
Single-cell analysis system:
-- MultiSample NanoDispenser (MSND) deposits nanoliter volumes of cells and reagents into the iCELL8 Chip in a temperature and humidity controlled environment
-- MSND is capable of dispensing 8 unique samples evenly across the chip
-- Full length single cell sequencing capacity