BAC-Recombineering Core

Cell Culture and Lines

Tissue Culture Facility

The CSC is a fully operational tissue culture facility capable of handling a large number of cell lines at any one point in time. The CSC currently has the capacity for 3600 frozen vials of cells with the ability to triple that capacity if necessary. To aid in cell counting for maintenance and plating a Beckman Coulter Vi-Cell XR is used which is capable of calculating cell concentration as well as cell viability. There are two biosafety cabinets in the CSC, one class A2 and one class B2. Four Class 100 incubators also provide ample storage for cell growth needs. A refrigerated centrifuge and an inverted microscope with fluorescence complete the setup in the tissue culture facility.

Cell Lines

The CSC is aiming to become a center for cancer cell line models within the Chicago area. The CSC currently holds 25 cancer cell lines with an emphasis on breast cancer lines. Ultimately, the CSC will hold representative lines for all known cancer types. The CSC is also collecting lines from other disease areas such as ophthalmology and lung disease.

Screening Reagents

The following screening resources are available to CSC users:

• RNAi libraries The CSC uses the Dharmacon genome-wide human siRNA library in house. Other vendors are also being explored for siRNA and shRNA libraries. These additional libraries will provide us with the ability to screen a larger number of cell lines and reduce “off-target” effects.
• Small compound libraries.

Chemical screens are performed in the CSC using libraries including:

• Chem Bridge Express-pick small molecule library of 50,000 molecules, eventually will house over 150,000 molecules from this library. [more info]
• NCI Cancer Compounds — 39 compounds from the NCI that are FDA approved for cancer treatment.
• Prestwick Chemical Library contains 1,120 small molecules, 90% being marketed drugs and 10% bioactive alkaloids or related substances, thus it presents the greatest possible degree of drug-likeliness. The active compounds were selected for their high chemical and pharmacological diversity as well as for their known bioavailability and safety in humans. The Prestwick Chemical Library was designed to reduce the risk of “low quality” hits, reduce the cost of the initial screening, and accelerate lead discovery. [more info]
• Natural products library contains over 5,000 plant and marine natural product extracts.

The Institute is also collaborating with medicinal chemists at the University of Illinois at Chicago to formulate more potent compounds for secondary screens.

Screening Fees

CSC staff will meet with the collaborating lab to assess the goals of the screen, assay details and readout, and feasibility of adapting the assay to a high-throughput format.

Following this initial consultation, Center staff will prepare a project cost estimate. They will estimate screening costs based on the assay type, readout, and estimated length of time the screen will take.

Note that the length of the screen will depend on setup time as well as the actual time to perform the screen. The CSC strongly recommends the use of CSC validated cell lines (list available upon request) in order to reduce the setup time and therefore reduce total project costs.

Lab Protocols

ENTERING THE CELLULAR SCREENING CENTER

Pre-Screening Guidelines for the CSC

Media Change Protocol

Bringing Materials Into CSC Protocol

Labware Washing in the CSC

Protocol for DAPI Staining

Protocol for Labeling Cryovials Before Banking Cell Lines in Liquid Nitrogen

Compound Screening Protocol

Biotek Washer ELx405 Day Maintenance Protocols

Beckman Coulter Vi-Cell Protocol

HDAC Inhibitor Protocol

Cell Banking Protocol

Renewing Reviving Cell Lines from Frozen Cryovials in the CSC

Combi Cassette Cleaning Protocol

Technology

All instruments are run on a consistent schedule which includes the ability to continue assays twenty-four hours a day. Remote access and two network video cameras allow for remote monitoring allowing the seamless completion of all assays as well as troubleshooting.

Robotic Deck

The CSC robotic deck includes a large workstation robot for sample prep and plate design which is integrated with 3 readers and 5 liquid handlers.

Thermo F3 Robotic Arm

This is the largest robotic arm available from Thermo and is named F3 for its capability of carrying loads up to 3 kg. The arm is placed on 3 meter rail with access to end table as well 900 mm reach. It has 6 degrees of freedom with 360 degree range of motion and reliable placement of +/- 0.05 mm. POLARA software allows scheduling of multiple assays using the arm.

Readers

There are 3 readers in the CSC enabling multiple different reads within a single screen. All readers are integrated with the Thermo F3 robotic arm.

Molecular Devices Analyst GT

Multimode reader for absorbance, luminescence, fluorescence intensity, fluorescence polarity, and time-resolved fluorescence resonance energy transfer (TR-FRET). This is the fastest reader on the deck but has greater assay cost than the other readers. Currently the CSC has filters for rhodamine and GFP.

Molecular Devices ImageXpress

Micro Automated fluorescent microscope with filters for DAPI, FITC, GFP, and TRITC. It has the slowest read time, especially if imaging entire well, but a lower cost per well than the other readers. It is integrated with environmental controls for time course experiments and produces large image files.

TTP Labtech Acumen Explorer

Laser-based (488 nm) fluorescent microplate cytometer enables whole plate reads in 10 minutes regardless of plate type. This has a large field of view and 2 PMTs per scan. Its output is generated as small CSV files and includes algorithms to detect fluorescence above background.

Liquid Handlers

All liquid handlers except the Tecan Freedom EVO 200 are integrated with the Thermo F3 robotic arm.

Tecan Freedom EVO 100

Includes an arm with eight tips, each of which can move independently to construct rearrays of RNAi master plates. Capable of dispensing to 96, 384, and 1536 well plates. The low volume capabilities of this instrument also allow for the dispensing of low volumes of precious reagents without significantly decreasing speed.

Tecan Freedom EVO 200

This all-in-one rearray and plate replication robot also includes an eight tip arm but due to its large size it can accommodate a 384 well head for plate replication as well as a plate manipulator arm for moving plates across its large deck.

PerkinElmer EP3

This a Universal pipetting platform with the unique capability that its 96 well and 384 well heads can be interchanged in thirty seconds without user intervention. This allows for rapid plate replication, whole plate transfers, and reagent additions. Its flexible gripper option allows for plate manipulation on the deck and its pipetting range is from 0.5 to 235 μl. It has a 16 position deck with some positions used for tip wash stations and tip load stations. Reagent auto-fill option is also present. Its strong interface allows for complete flexibility.

Thermo Electron MultiDrop Combis

Two of these instruments are integral for bulk reagent and cell dispensing. They are an excellent combination of robust design and versatility, dispensing 0.5-2500 μl to plates from 6 to 1536 wells and from heights of 5 to 50 mm. 100 μl can be dispensed into a 96 well plate in 10 seconds. They have a new updated design with an LCD interface and are highly robust instruments with low CVs, high precision, and high throughput speeds.

BioTek ELx405 Select UCWVS

This plate washer is specifically designed for cell work. The “low-flow” capability of the instrument washes cells without disturbing their adherence to plates and the ultrasonic washing helps ensure sterile manifolds. Media changes and washes would normally be a bottleneck in the process; this instrument eliminates this bottleneck. It is useful for ELISAs as well, multiple washes are done quickly and easily.

Thermo Cytomat 6001

This incubator has a 189 plate capacity and temperature range of +10-50 °C. It is located below the CSC deck to optimize deck space.

Thermo 7 Station Delidder

The 7 station delidder allows 7 plates to be active at any one time.

Thermo 200 Position Carousel

provides nests for 200 standard well microplates.

Thermo Scientific Matrix PlateMate

Plus is a high throughput 96- or 384-channel automated pipetting system.

Microscan MS3 Barcode Reader

Organization is extremely important in the high throughput environment of the CSC so a 1-D barcode reader is fully integrated into all screens and all plates are barcoded for time logs as well as tracking.

Laboratory Space

CSC Laboratory Specifications

The CSC is housed in a 900 square foot laboratory which is designed as an adaptable space for a broad range of screening assays. The CSC includes certified Class 10,000, 1000 and 100 space in which the air is filtered to reduce the number of particles to 10,000, 1000 or 100 per cubic meter respectively. This process ensures a high degree of confidence in assays due to the vastly reduced risk of contamination in this environment. [high-res image]

Class 10,000 Space

Class 10,000 space includes an anteroom in which CSC staff and users change into protective clothing before entering the main facility. [high-res image]

Class 1000 Space

Class 1000 space includes:

• Large amount of storage / general use space as well as the tissue culture room
• Two -80° freezers
• One -20° freezer
• One 4° refrigerator
• Four CO2 incubators
• Two biosafety cabinets: one A2, one B2
• Inverted microscope in tissue culture room
• Beckman Coulter Vi-Cell XR for automated cell counting

[high-res image]

Class 100 Space

Class 100 space includes two areas, one designed for the Tecan Freedom EVO 200 and another designed for Thermo F3 arm system. Both areas are protected by soft wall curtains to ensure Class 100 space. [high-res image]

High-Throughput Genome Analysis Core

The High-throughput Genome Analysis Core (HGAC) is a state-of-the-art facility providing resources and services for ultra-high-throughput sequencing and large-scale microarray processing. Operated by the Institute for Genomics and Systems Biology, the HGAC facility is available to University of Chicago and Argonne National Laboratory investigators and their collaborators.

Services

The HGAC facility offers the following services:

• Solexa and 454 sequencing
• High-throughput microarray processing (expression and tiling arrays)

Technologies

Current technologies at HGAC include the following:

• 454 sequencer (Roche GS-FLX). The 454 generates up to ~1,000,000 single reads with an average read length of 400 bases (~400 Mbp per instrument run). Current 454 applications include de novo sequencing, resequencing; discovery of small RNAs and detection of their relative abundances, detection of low-frequency (<5%) somatic mutations, uncovering the diversity in metagenomic samples, and transcriptome sequencing.
• Illumina Solexa GAII.  The HGAC facility houses four Illumina Genome Analyzers.  Each Solexa GAII instrument generates 10-25 million reads at 18-100 bp per lane with eight lanes available per run.  Up to 40 billion bp of usable data can be generated on each Solexa GAIIx instrument per run when run with the Illumina Genome Analyzer Paired-End Module.  Applications on the Solexa platform include: genome sequencing and resequencing; transcriptome sequencing; amplicon sequencing; and ChIP-seq.
• Microarray
• Tecan HS 4800. The Tecan HS 4800 Pro Hybridization Station can process up to 12 microarrays simultaneously.
• Agilent SureHyb. Our SureHyb pipeline can process 48 slides simultaneously.

Sample Submission Guidelines

Requirements

The quality and quantity of the DNA/RNA sample are critical to the success of these procedures. Any contamination in the starting material will be directly reflected in the output library.

Ideally, the DNA/RNA sample should be checked to ensure the material is derived from the target organism and contains no other contaminating DNA. At a minimum, the DNA sample should meet the following criteria:

• DNA should not be degraded, and should contain no particulate matter
• OD260/280 ratio should be approximately 1.8
• Concentration should be 5 ng/ul or above, in TE or water (0.5 ng/ul for cloned or PCR-generated targets)
• Because DNA quantitation using OD260 is variable and dependent upon DNA purity, the input DNA concentration should be verified by fluorometry (PicoGreen/Ribogreen-based assay; Invitrogen Qubit) or densitometry (e.g., after gel electrophoresis on a 1 - 2% agarose gel using a DNA Mass Ladder).

Samples can be in water or TE. Samples can be shipped in a 1.5mL labeled tube, microtiter plate, or boxed. Samples and containers must be clearly labeled. If submitting unboxed 1.5mL tubes sample tubes can be sealed with parafilm, placed in a 50mL screw-top falcon tube packed with KimWipe, and shipped frozen on dry ice. Please include plenty of dry ice with your samples. Please include concentration, 260/280 readings, and a gel image for each submitted sample.

Pricing

Illumina GAII (Solexa)
* 1 Solexa cycle = 1bp
* Solexa lane run for 36-cycles typically generates 200-1000Mbp (5-25 million reads per lane)
* 7 lanes available for user samples per run; 1 lane used as control (all 7 lanes do not have to be from same user)
* Illumina currently offers 12 different library barcode indexes allowing 12 libraries to be run simultaneously in a single lane.
* Read length currently supported on Solexa: 18-100bp

36-cycle Solexa run without library preparation = $1,000.35 per lane
46-cycle Solexa run without library preparation = $1,172.34 per lane
56-cycle Solexa run without library preparation = $1,256.14 per lane
76-cycle Solexa run without library preparation = $1,511.93 per lane
100-cycles Solexa run without library preparation = $2,023.50 per lane
Standard Solexa DNAseq, RNAseq, or Paired-end library preparation = $425.40 per library
Standard Solexa ChIP-seq library preparation = $362.04 per library
2x36-cycle Solexa Paired-end run without library preparation = $1,881.24 per lane
2x54-cycle Solexa Paired-end run without library preparation = $2,392.81 per lane
2x76-cycle Solexa Paired-end run without library preparation = $2,610.05 per lane
2x100-cycle Solexa Paired-end run without library preparation = $3,338.86 per lane

Roche GS-FLX (454)
* Instrument can be run using either LR70 or XLR70 (Titanium) kits
* Only the shotgun DNAseq kit is currently available for XLR70 chemistry (Amplicon and Paired-end kits available late 2009)
* 454 Plate can be configured with 2, 4, 8, or 16-regions gaskets; 2-region gasket gives maximum Mbp output
* 12 Library Multiplex Identifier (MID) barcodes are available to allow for up to 12 libraries to be run per region.
* LR70 2-region output = ~400,000 reads with ~250bp mean fragment length (Total output = ~100Mbp)
* XLR70 2-region output = ~1,000,000 reads with ~400bp mean fragment length (Total output = ~400Mbp)
LR70 Standard 454 shotgun DNAseq library preparation = $539.74 per library
LR70 454 Sequencing Run = $13,390.35
XLR70 (Titanium) standard 454 shotgun DNAseq or cDNA library preparation = $496.77
XLR70 (Titanium) 454 Sequencing Run = $13,073.29

Microarray
Current slide processing full costs below:
- 1 array/slide hyb only (Tecan):  $154.91
- 4 array/slide hyb only (Tecan):  $154.91
- 1 array/slide hyb only (Manual):  $116.57
- 4 array/slide hyb only (Manual):  $116.57
- GE dual-color labeling:  $385.23
- GE single-color labeling:  $212.80
- ChIP-chip dual-color labeling: $255.41

Shipping Address

ATTN: IGSB HGAC Lab
Argonne National Laboratory
9700 South Cass Avenue
Building 202 Room A346
Argonne, IL 60439-4803
Phone: (630) 252-3993

Technology

Roche GS-FLX (454) Next Generation Sequencing

The Roche Genome Sequencer FLX System (GS-FLX or “454”) moves DNA sequencing to a chip based platform dramatically reducing the work and price required for genome level sequencing projects. This is performed by simplifying the up front work required to generate and amplify a genomic library and by massive parallel sequencing on a fiber optic chip.  The 454 sequencer currently generates 1,000,000 reads per run at a current mean read length of 400-500 bp (~400 Mbp output per instrument run).  454 applications include: de novo sequencing; re-sequencing; discovery of small RNAs and detection of their relative abundances; detection of low-frequency (<5%) somatic mutations; uncovering the diversity in metagenomic samples; amplicon sequencing; and transcriptome sequencing.

• Amplicon Library Preparation Method Manual [PDF]
• Amplicon Fusion Primer Design Guidelines for GS FLX Titanium Series Lib-A Chemistry [PDF]

Solexa Next Generation Sequencing

Like the Roche GS-FLX sequencer the Solexa Genome Analyzer (GA) moves DNA sequencing to a chip based platform dramatically reducing the work and price required for genome level sequencing projects. An entire genomic library is amplified for sequencing simultaneously on a chip via bridge-PCR and then each clonally amplified product (cluster on the chip) is sequenced by synthesis on the Solexa GA by massive parallel sequencing across the entire chip (Solexa ‘flow cell’). During sequencing each terminating base is labeled with one of four fluorophores allowing for the reading of one base per sequencing cycle per cluster. Each Solexa GAII instrument generates 10-25 million reads at 18-100 bp per lane with eight lanes available per run.  Up to 40 billion bp of usable data can be generated on each Solexa GAIIx instrument per run when run with the Illumina Genome Analyzer Paired-End Module.  Applications on the Solexa platform include: genome sequencing and resequencing; transcriptome sequencing; amplicon sequencing; and ChIP-seq.

High-throughput Microarray Processing

The IGSB HGAC performs tiling and expression microarray hybridizations using the Tecan HS 4800 PRO Hybridization Station and the Agilent manual SureHyb protocol. Our facility has the current capacity to process 12 slides simultaneously on Tecan and 48 slides simultaneously following Agilent’s SureHyb protocol. To date we have processed Agilent arrays in a high-throughput format following Agilent’s Gene Expression or ChIP-seq protocols. We have two scanners, a single slide Axon 4000B 5um scanner and a C-version Agilent Autoloader scanner capable of scanning up to 48-slides at a maximum resolution of 2um.

Staff

Staff

The staff of the CSC have the express mandate to assist Chicago investigators in adapting cell-based assays for high throughput screening and to perform that screening at cost. Based on their previous experience, this team advises users on the best assay systems to fit their needs (considering factors such as assay robustness, economy, assay speed). The team then works with the user to import their assay into the facility and adapt it for high throughput screening. Finally, the CSC team and user conduct the screen, and CSC staff aid the user in primary analysis of the resulting data.

Screening Guidelines

Before utilizing the CSC there are several steps that must be completed to insure quality, timely screens.

1. Contact is made between collaborating labs and the CSC. At this point the discussions will entail topics such as goals of the screen, assay details and readout, and feasibility of the assay adapted to a screen.
2. A representative from the collaborating lab is assigned to act as a liaison between the CSC and the lab. This representative is responsible for performing assay development, maintaining communication, and assisting with troubleshooting for the screen. This person will also be assigned a representative from the CSC who will act as the project manager for the screen.
3. The assay must be presented in 96 or 384 well format in plates that are SBS compliant and automation friendly. The CSC is able to provide recommendations for plate types based upon assay and readout parameters.
4. The CSC offers three different readers: the Molecular Devices Analyst GT, the Molecular Devices ImageXpress Micro, and the TTP Labtech Acumen Explorer. The assay readout must be compatible with one of these readers. Once assay development has been completed in the collaborating lab, the assay must be validated in the CSC; most importantly, using the reader(s) in the CSC.
5. The assay will be adapted and validated to use the automation in the CSC. During this time, the representative from the collaborating lab will assist with any assay troubleshooting that may arise.
6. Screen is performed in the CSC.

Cellular Screening Center (CSC)

The Institute’s Cellular Screening Center (CSC) houses a state-of-the art high throughput cellular screening facility which opened in 2007. Support from the Chicago Biomedical Consortium and the University of Chicago Biological Sciences Division enabled the creation of CSC.

The CSC facilitates drug discovery and development by defining the complex genetics underlying disease, isolating chemical compounds that affect specific cellular activities, and developing potential therapeutics to target disease pathways.

Automation is present for all aspects of the screening process. Without automation a 300 plate screen would take as long as 2 months. With the automation present in the CSC the same screen can be performed in as little as 2 days.

CSC is available to a broad network of Chicago area investigators on a collaborative basis established through internal and external funding. The Center has attracted a significant number of collaborations with area scientists in fields as diverse as cancer biology, diabetes and ophthalmology.

Overview

IGSB features state-of-the-art core technologies at the University of Chicago and Argonne National Laboratory. These facilities support efforts to define the complex genetics underlying disease and the development of potential therapeutics to target disease pathways.

Current and upcoming facilities include:

Celluar Screening Center (CSC)

• High-throughput RNAi and chemical genetic screening

High-throughput Genome Analysis Core (HGAC)

• Tiling and expression arrays for large-scale experiments
• Sequencing using the speed and cost efficiency of the latest technologies including Solexa and 454

Micro-Western Array Core (MWA)

• The Micro-Western Array (MWA) Core is a technological initiative designed to provide a quantitative platform for studies of abundance and modification of pre-selected protein targets.

BAC-Recombineering Core (BRC)

• The BAC-Recombineering Core can provide investigators affiliated with CBC schools the capability to dynamically image fluorescently tagged proteins in model organisms and measure spatiotemporal expression of protein of interest.

Any questions regarding the Institute’s facilities should be addressed to Dr. Aharon Solomon, IGSB Research Director email: .(JavaScript must be enabled to view this email address) (773) 834-3882