Yale HTCB Executive Summary

Outline

•! Mission Statement and Center Refocusing •! Timelines •! Personnel •! Resources •! Projects •! Challenges and Solutions •! Summary and Future Directions

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Mission Statement

•! The goal of the Yale Center for High Throughput Cell Biology is to provide the expertise, technology, and resources needed to advance the fields of molecular and cellular biology. Our current focus on RNAi screening underscores our commitment to the principle that probing the details of cell signaling by modulating gene expression will elucidate the mechanisms for life, disease, and death.

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Refocusing Center Efforts

•! "The Center will develop and provide assays and related services for RNAi, cDNA, Chemical Library and other high throughput screens that can be used to probe basic cellular mechanisms and, in due course, the function of human disease genes."

•! "The goal of the Yale Center for High Throughput Cell Biology is to provide the expertise, technology, and resources needed to advance the fields of molecular and cellular biology. Our current focus on RNAi screening underscores our commitment to the principle that probing the details of cell signaling by modulating gene expression will elucidate the mechanisms for life, disease, and death."

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Timelines

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May-08 June-09

Milestone April 1st:

Technological infrastruture

Milestone June 30th:

10 screens completed @10,000 genes per

screen

Timeline: Team-building

Lars Branden Director

Adrian Poffenberger Informatics

Marie-Aude Guie Michael Richo Phil Williams

Michael Wyler Assay Development / Implementation / Imaging

Deborah Smith Anthony Raffo Yan Song

Ashima Bhan Leena Kuruvilla

Susanne Hasse

Steven Berman Operations

Lori Ortoleva-Donnelly HTS

Jason Ignatius

Trisha D’Errico Administration

Laurie Tomei

January-09

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May-08 January-09 June-09

Milestone April 1st:

Technological infrastruture

Milestone June 30th:

10 screens completed @10,000 genes per

screen

Ordering Cell

Culture Equipment

Assemble Cell Culture work area

Issues with computers, campus mail, FedEx, shuttle, Corporate Express, Accuship, stock room purchases,

p-card

Renovations of HTCB space

Scope of work to be done Project Budget $300,000 – Project

Cost $260,000

Scheduled work orders for services still required

to get HTCB up and running

Purchasing process, invoicing, ISP, CDA and Contract Template

Timeline: Renovation and Yale Integration

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May-08 January-09 June-09

PLATE SEAL REMOVAL SYSTEM #1 and 2

STORAGE SYSTEM N3300 EXPRESS

APPLICATION SERVER ORACLE DATABASE SERVER

INCUBATOR HERACELL CO2 #1 and 2

Opera

Biorad Imaging system

#2 Enclosure

#3 Enclosure

CELLULAR SCREENING WORKSTATION #2 and 3

Immunostaining WORKSTATION #4

Milestone April 1st:

Technological infrastruture

Milestone June 30th:

10 screens completed @10,000 genes per

screen

UltraVIEW

DISK DRIVE SUBSYSTEMS

FREEZER #1-6 FREEZER CONTROL RATE

NITROGEN FREEZER ROBOT #1

Timeline: Equipment

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Dimaio Genetic Requirements of Senescence

Corson / Crews Triptolide Assay

Sessa LDL Uptake

Chapin PC-1 tail trafficking

Shadel Mitochondria Dynamics

Pober TNF

Saltzman Glioma stem cells

Fernandez-Hernando Cholesterol Trafficking

Mader Matrix degeneration in breast cancer

May-08 June-09

Milestone April 1st:

Technological infrastruture

Milestone June 30th:

10 screens completed @10,000 genes per

screen

Timeline: Projects

Schepartz Cell-Penetrating Miniature

Protein

Pritchard Transcription factor K-D

Scott Small APP-BACE

Oyagen EVVVHR Quenched FRET

Scott Small VPS35

Sigma Antibodies and Assays

HTCB NFAT Adeno Assay

HTCB NF!B validation assay

HTCB AP1 Adeno Assay

January-09

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Personnel

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Organizational Chart

Resources

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Tecan Evo Freedom 200 Highly advanced, fully automated liquid and plate handling platforms capable of processing 1536-well plates. Our platforms can deliver liquid volumes from 10 nanoliters to 125 microliters and can process over 200,000 wells per day. Barcoded screening plates allow digital plate tracking and real-time data streaming to our enterprise-scale sample, plate, and results database.

Perkin-Elmer Opera High throughput imaging system for high content sub-cellular imaging and analysis of more than 70,000 samples/day in three colors.

Infinite 1000M Plate readers are integrated with the automation platforms to provide a broad spectrum of detection capabilities including fluorescence, luminescence, absorbance, and FRET.

Infrastructure

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Infrastructure •! Scalable hardware infrastructure housed within a modern industrial-scale data center. •! Databases and data processing applications primarily utilize IBM x3850, IBM x3650, and Mac Xserves in

addition to other systems. •! Computational projects are supported internally via our 24-CPU Linux cluster. For larger projects we utilize the

Yale High Performance Computing Cluster, which currently provides 1000 CPUs. •! Primary storage is managed by an IBM n3300 SAN device. Hard disk space is presently over 10TB and is

quickly scalable to 80 TB on existing controllers and equipment. •! Backups are performed on an IBM TS3200 tape device capable of archiving over 75 TB per batch, and backups

are managed using an IBM x336 server. Long-term archiving is performed on a Blu-Ray optical disc jukebox. •! Network infrastructure provides 1 Gb/s connectivity throughout the Yale West Campus facilities and 10 Gb/s

fiber to the Internet. •! Our primary scientific applications include ActivityBase suite (IDBS); Pipeline Pilot (Accelrys); MetaCore

(GeneGo); and Acapella (PerkinElmer) for high content image analysis and feature extraction. •! Fully validated industry-scale disaster recovery plan.

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Dharmacon siGenome Human siRNA Library

Total: 21,122 genes

Druggable Genome: 5906 genes

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Projects

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Projects

•! Free-screens for Yale Investigators

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Yale Free Screens

•! October 3, 2008 – Introductory email sent to Yale community along with request for proposals for free screens.

•! October 20, 2008 – Deadline for free-screen applications.

•! Response rate: 37 applications received for 10 free screens out of 9,562 email solicitations sent.

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Initial Screen Purpose: To find Binding Partners of PKD2 Involved in Triggering Cell Death.

Model System – Triptolide is toxic to Hela Cells. Biochemical studies demonstrated

that triptolide binds to PKD2, a calcium channel present in the plasma membrane.

Assay – Treat cells with triptolide and 24h later, measure cytoplasmic ATP levels.

Problem – Positive controls did not work as expected, i.e., siRNA to PKD2 and

caspase-3 did not block reduction in ATP at 24h.

Final Screen Purpose: Screen for targets related to triptolide toxicity.

Assay -- Treat cells with triptolide and use luminescent assay for caspase-3

activation at 18h.

Corson – Triptolide P.I., Craig Crews

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Projects

•! Clients beyond Yale University

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Oyagen Assay-240K Compound Screen Harold Smith P.I., Oyagen, Rochester, NY

Assay Description –! VIF, an HIV encoded

protein, forms a dimer that allows for infection by disrupting antiviral activity of a human enzyme, APOBEC

Assay Principle –! Transfect with EVV:VHR

and look for compounds that prevent VIF dimerization

–! Quenching assay EVV EVV:VHR

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Sigma-Aldrich Antibody Validation

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Genetics of Transcription Factor Targets Jonathan Pritchard, Yoav Gilad at Univ of Chicago

HapMap Project: International Effort to

study patterns of human genetic variation.

Nigerian (Yoruba) European

Japanese

Chinese

Project: Target Transcription Factor genes with shRNA in a lentivirus Vector. Prepare total RNA for qPCR and Microarray.

Project Phase 1: One Cell line: B-lymphocytes (EBV transformed) (Yoruba)

50 Targets: 48 TF genes plus 2 Controls

5 shRNAs per gene In Triplicate

750 RNA

Isolations 24

Genetics of Transcription Factor Targets Jonathan Pritchard, Yoav Gilad at Univ of Chicago

HapMap Project: International Effort to

study patterns of human genetic variation.

Nigerian (Yoruba) European

Japanese

Chinese

Project: Target Transcription Factor genes with shRNA in a lentivirus Vector. Prepare total RNA for qPCR and Microarray.

Project Phase 2: Five Cell Lines

2000 Targets: 1998 TF genes plus 2 Controls

5 shRNAs per gene In Triplicate

150,000 RNA

Isolations 25

Projects

•! Informatics Projects

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Informatics Projects •! Protein Half-life prediction •! Gene Profiler •! siRNA off-target prediction •! Mouse/human siRNA target gene overlap •! Parallelized image analysis •! Secure web-based client data access •! Fully integrated image management

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Hypothetical diagram of molecular turnover time comparison

siRNA levels decline

from dilution and degradation

Protein level declines from

normal turnover with non-replacement due to silencing

Incubation Time

after transfection

Concen

tration

Optimum time window

for experimental measurement

Protein level

returns to normal

Transfection started

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NFkB Signaling Pathway

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Highest average interaction score

Shortest path length

Highest common gene count

Multiple algorithms

Assay Plate: hits in red

Gene Profiler