Rhode Island Genomics and Sequencing Center David R. Nelson, Paul W. Johnson and Janet A. Atoyan

Department of Cell and Molecular Biology, 352 Center for Biotechnology and Life Sciences, University of Rhode Island, Kingston, RI 02881

Introduction

The Rhode Island Genomics and Sequencing Center (RIGSC) was established to provide technical and analytical

support for molecular biology and genomics research at the University of Rhode Island and all RI-EPSCoR

institutions. The mission of the RIGSC is to facilitate interdisciplinary genomics research and undergraduate and

graduate student training opportunities by providing researchers access to cutting-edge technologies in the field of

genomics. The RIGSC offers services in robotic sample preparation, DNA sequencing (Sanger and Next Generation),

fragment analysis and quantitative-PCR. We also provide imaging services using transmitted light, epifluorescence,

and laser scanning confocal microscopy, as well as cryostat sectioning of frozen specimens. The RIGSC is available to

students, staff, and faculty at URI, as well as RI-EPSCoR researchers. Detailed information on sample preparation,

submission instructions, equipment use and any fees is available on our website at web.uri.edu/gsc/.

Applied Biosystems 3130xl & 3500xl Genetic Analyzers

These instruments are fluorescence-based genetic analysis

systems using capillary electrophoresis for DNA sequencing

(Sanger method) and fragment analysis; fully automated from

polymer loading/replacement to DNA separation, detection, and

data analysis enabling 24-hour unattended operation. Both

instruments typically generate very high quality sequences with

read lengths greater than 900 bases.

Specifications:

• Auto loading of samples performed from two micro-titer plates;

either 96- or 384-well format.

• Arrays with either16 or 24 capillaries and 50 cm length.

• Automated delivery system for POP-7 polymer.

• Argon laser with primary excitations at 488 and 515 nm.

• Detection of up to 5 dyes for sequencing and fragment

analysis.

• CCD detection technology and spectrograph for color

separation.

• Software for DNA sequencing/base calling (Sequencing

Analysis v 6.0) and for fragment analysis of microsatellites, SNP,

AFLP and LOH (GeneMapper v 5.0) as well as comparative

sequencing, mutation detection, and detection of heterozygote

insertions and deletions (SeqScape v 2.5)

Eppendorf epMotion 5075 VAC

The Eppendorf epMotion 5075 VAC automated pipetting workstation enables fast and reproducible liquid

handling for a variety of applications. The 5075 VAC system can pipette from single tubes, plates or reservoirs to

24-, 96- and 384-well plate formats as well as handling specified labware with a gripper tool. Applications include

diverse methods such as nucleic acid purification, PCR setup, cleanup of sequencing reactions, etc. An integrated

vacuum station can also be used for high throughput nucleic acid isolation and purification.

Specifications:

• Workstation has 12 rack positions in 96/384-well standard

plate formats

• Precise, reproducible dispensing from 1 µl to 1,000 µl

• Labware handling with gripper tool

• Optical Sensor for high-precision and contact-free liquid

level sensing as well as recognition of tips and labware

• epMotion Editor software package for generating and

editing methods as well as printing and archiving program

processes

• Labware database with more than 300 validated entries can

be used to individually compile the system configuration in

relation to plates, tubes and liquid classes.

Thermal Cyclers

Eppendorf Mastercycler ep Gradient S thermal cyclers (2) in

96-well format for PCR. Peltier elements control the

temperature of the silver coated block with extremely fast

heating and cooling rates.

Specifications:

• Intuitive graphic programming

• Heated lid with integrated ESP technology (Electronic Sample

Protection)

• Highest temperature speed due to silver block: up to 6° C/sec

• Gradient block with SteadySlope™ technology allows freely

programmable temperature gradient over 12 rows. Availability

of a gradient range from 1° to 24° C enables easy optimization

of PCR components.

Quantitative PCR Systems

Quantitative PCR (QPCR) allows researchers to quickly and easily quantify nucleic acids for studying gene

expression, mutational analysis, disease state, and gene dosage. QPCR measures PCR product accumulation

during the exponential phase of the reaction and before amplification becomes vulnerable to limiting reagents

and cycling variability. Fluorescent QPCR data provides accurate information on initial starting copy number.

Using QPCR, amplification and detection are combined in a single step and in a single closed tube. This

eliminates the need for numerous post-PCR manual steps, and reduces the possibility of introducing variability

or laboratory contamination. The GSC offers two platforms with many chemistries including SYBR Green dye

and fluorogenic probes, detecting four to five different dyes simultaneously. These systems support both

quantitative and plate read experiments.

Agilent/Stratagene Mx3005P

Specifications:

• Excitation source: Quartz-tungsten halogen lamp

• Excitation range: 350 to 750 nm

• Detector: Photomultiplier tube (PMT)

• Detection range: 350 to 830 nm

• Filters: FAM™/SYBR® Green I (492nm-516nm),

HEX™/JOE™/VIC™ (535nm-555nm), ROX™/Texas

Red® (585nm-610nm), Cy5™ (635nm-665nm) and Cy3

(545-568)

• Thermal system: Peltier-based, 96-well block thermal

cycling system; temperature range 25–99°C

• Sample format: Standard 96-well plates, 8-strip tubes, 200

µl tubes, optimized for 25µl reactions

• Linear dynamic range: 10 orders of magnitude, standard

curve method

• Temperature uniformity: +/- 0.25 ˚C at 72 ˚C; ramp rate:

up to 2.5 ˚C/second

Zeiss Axio Imager M2 Light Microscope and AxioCam HRc Digital Camera

The RIGSC's imaging systems utilize a ZEISS Axio Imager M2 upright microscope that is equipped for

transmitted light microscopy with brightfield, phase-contrast, and differential-interference contrast (DIC)

methods. The Axio Imager M2 is also set up for epifluorescence microscopy using an HXP 120 metal halide

lamp and filter sets for DAPI, FITC, Rhodamine and Propidium Iodide excitation / emission. Almost all

functions of the Axio Imager M2 are motorized and can be operated through a touch-sensitive TFT display.

The Axio Imager M2 is also equipped with a motorized stage to allow for automated acquisition at multiple

locations and tiled montage images with correlated image stitching.

Specifications:

• Sample size: 1.0 to 20.0 µl

• Single sample well with measurement in 3 sec.

• Sensitivity of assay kits:

RNA broad range kit = 20-1,000 ng

RNA kit = 5-100 ng

ssDNA kit = 1-200 ng

dsDNA broad range kit = 2-1,000 ng

dsDNA high sensitivity kit = 0.2-100 ng

ZEISS AxioCam HRc digital camera is a high

resolution color digital camera that can be used at

different resolutions depending on the application: from

1388 x 1040 up to 4164 x 3120, corresponding to 13

megapixels per color channel. With two different read-

out speeds (12.5 MHz and 25 MHz) the HRc is versatile

in terms of the range of applications it can perform. With

the fast read-out mode of 25 MHz, live images of

between 12 images/sec (full resolution) and 33

images/sec (reduced resolution) can be achieved. The

extremely high sensitivity of the large 1.7 cm sensor, an

outstanding signal to noise ratio and Peltier cooling for

long exposure times allow the HRc to make high-quality

imaging possible.

Zeiss LSM 700 Scanning Confocal Imaging System

The LSM 700 laser scanning confocal module is another component of the Zeiss Axio Imager 2 light

microscope. This enhanced imaging capability provides true confocal or laser scanning technology for the

examination of cells and tissues that are labeled with one or more fluorescent probes. Confocal imaging

permits greater resolution of thick specimens and localization of organelles and gene expression labeled by

very specific fluorescent probes. The LSM 700 optics are designed for high transmission and diffraction-

limited imaging over the spectral range 350–800 nm. All LED lasers are fiber coupled to the scan head and

the optical fibers are directly coupled to the laser output windows for long-term stability.

LSM 700 Laser Specifications:

• Violet, 405 nm (DAPI), 5 mW

• Blue, 488 nm (FITC), 10 mW

• Yellow, 555 nm (Cy3), 10 mW

• Red, 639 nm (Cy5), 5 mW

Acknowledgement of Support

The RIGSC acknowledges the support of the National Science Foundation (MRI

Grant No. DBI-0215393 and EPSCoR Grants No. 0554548 and EPS-1004057), the

US Department of Agriculture (Grants No. 2002-34438-12688 and No. 2003-34438-

13111), and the University of Rhode Island.

Contact Information

The RIGSC website is http://web.uri.edu/gsc/. Please see our Services menu

for detailed information on sample preparation, submission instructions,

equipment use and the current fees structure. The Equipment menu provides a

list of equipment and specifications. Please consult the Calendar for equipment

and service availability and to make a reservation (approved users only).

Training is required for all users prior to operating equipment in the RIGSC.

Contact the RIGSC for additional information or to schedule a training session

on any piece of equipment: 401-874-5919; pwj419@uri.edu, jatoyan@uri.edu

or gsc@etal.uri.edu.

Covaris S220 High Performance Ultrasonicator

Specifications:

• Sample volumes from 50 µl to 10 ml

• One sample per run with times of 50-900 seconds

• Average power: 100 watts

• Sample vessel: microTUBE with AFA fiber (< 130 µl

volume) for shearing 100-1,500 bp sized DNA

fragments; miniTUBE (< 200 µl volume) for generating

sheared DNA of 2-5 Kb size.

Illumina MiSeq Next Generation Sequencer

The MiSeq is an integrated instrument that performs clonal amplification, genomic DNA sequencing, and data

analysis with base calling, alignment, variant calling, and reporting in a single run. The MiSeq benchtop

instrument utilizes a double-sided, single-lane flow cell and reagent cartridge supplied in kit form. Sequencing

is performed by recording the synthesis of DNA strands in clusters of sample templates attached to the flow

cell. Each newly attached base liberates a fluorescent dye that is excited by diode lasers (530 & 660 nm) and

imaged using two digital cameras. Sequential interrogation of bases allows for the flexible adjustment of read

length during a run. Up to 96 samples may be sequenced in a single run with DNA libraries prepared with

indexed or bar-coded adapters. The capacities of the single lane flow cell are shown in the following table.

Read Length

(base pairs)

Run Time

(hours)

Data Output

(bases)

1 x 35 ~4 600 Mb

2 x 25 ~5 800 Mb

2 x 75 ~24 3.7 Gb

2 x 150 ~24 5.0 Gb

2 x 250 ~35 8.0 Gb

2 x 300 ~65 15 Gb

The Covaris S220 provides automated acoustic disruption and homogenization of cells and tissue samples using

focused ultrasonic energy at 500 KHz. DNA fragments are generated in highly reproducible size ranges down to

100 bp. Other applications of the S220 include RNA and protein extraction, chromatin fragmentation and the

lysis of cells, spores and organelles.

Agilent 2100 BioAnalyzer

The Agilent 2100 Bioanalyzer offers fast and automated separation,

sizing and quantification of DNA, RNA and proteins by miniaturized

Lab-on-a-Chip electrophoresis. All results are available as digital data in

30-40 minutes for 10 to 12 samples, depending on the assay. Qualitative

and quantitative results are provided in a single run using a 1 – 5 µl

sample volume. • DNA Solutions: Four kits are available for sizing and quantification

of PCR fragments and restriction digests from 25 to 12,000 bp in size.

The kits are DNA 1000, DNA 7500, DNA 12000 and High Sensitivity.

• RNA Solutions: Three kits are available for the analysis and

quantification of messenger and total RNA in 50 pg to 500 ng per µl

concentration. These kits are the RNA 6000 Pico, RNA 6000 Nano and

Small RNA.

Wafergen Apollo 324 System

The Apollo 324 is an automated liquid handling instrument dedicated to the preparation of sample libraries

for Next Generation Sequencing. This system uses Wafergen reagent kits to prepare DNA and RNA libraries

designed for the major NGS platforms. Throughput ranges from 1-8 to 32 samples per run. The Apollo 324

performs the following tasks: Isolate and purify nucleic acids, fragment and repair DNA, A-tail addition

(when needed), adapter ligation, and size selection with clean up using a bead-based method

Specifications:

• Throughput: 1 to 8 samples in 3.5 hours or less; 32

samples per day (64 with overnight run).

• Performs in-tip library reactions and magnetic bead-

based cleanups to eliminate DNA cross

contamination.

• Deck has dual 96-well blocks with heating and

cooling to maintain enzyme stability for optimal

performance.

• Input material: ≥ 100 ng genomic and cDNA; ≥ 0.5

ng ChIP DNA.

• Multiple built in protocols are accessed through

touch screen operation.

Roche LightCycler 480

Specifications:

• Reaction volumes: 10 µl - 100 µl (96-well)

• Temperature control: Peltier-based

heating/cooling from 37° C - 95° C

• Heating rate: 96-well block: 4.4° C/sec

• Cooling rate: 96-well block: 2.2° C/sec

• Excitation source: Xenon Lamp (430-630 nm)

• Detector: Cooled monochrome CCD camera

(1024 x 1344 pixels)

• Filters: Excitation: 440, 465, 498, 533, & 618

nm; Detection: 488, 510, 580, 610, 640, & 660 nm

NanoDrop 8000 Spectrophotometer

The NanoDrop 8000 spectrophotometer is a full-spectrum (220-750 nm) instrument that accurately measures

up to eight individual 1 µl samples simultaneously. The software allows the user to measure samples using

either the full 8-position mode or a convenient single-sample mode.

Applications:

• Nucleic acid concentration and purity of samples

up to 3700 ng/µl dsDNA without dilution

• Fluorescent dye labeling density of nucleic acid

microarray samples

• Purified protein analysis (A280 nm) up to 100

mg/ml (BSA)

• Bradford Assay analysis of protein

• BCA Assay analysis of protein

• Lowry Assay analysis of protein

• Pierce Protein 660 nm analysis

• Cell density measurements

Invitrogen Qubit Fluorometer

The Qubit fluorometer uses fluorescent dyes bound to specific molecules in order to quantify DNA, RNA

and proteins. Samples with concentrations as low as 10 pg/µl of DNA and 12.5 µg/ml of protein may be

accurately and reliably quantified.

Sage Science BluePippin

The BluePippin is a preparative electrophoresis platform that separates and extracts DNA fragments. Using

pre-cast gel cassettes, DNA is automatically collected in buffer according to software-input size ranges. The

BluePippin features pulsed-field electrophoresis capabilities that enable accurate collection of kilobase-

sized fragments. Cassettes are available for DNA size selections between 50 bp and 50 kb.

Specifications:

• Three types of gel cassettes: 0.75%, 1.50%, & 3.0% agarose

• Cassette capacity: 4 sample lanes and 1 size standard lane

• Load capacity: up to 10 µg with 200 ng optical sensitivity

• Pulsed-field electrophoresis collects fractions up to 50 kb

• Individual sample channels with collection chambers

eliminate cross-contamination

• Automated collection of PCR bands or restriction fragments

• Provides narrow fragment size distributions

AirClean Systems 600 PCR Workstation

Combination workstation provides an ISO 5 (FS209E) Class 100 clean air environment and UV light

sterilization for optimal protection from sample contamination. Raising the bi-fold sash turns off the UV

light and enables the blower during use.

Specifications:

• UVTect™ microprocessor controller

• Class 100 (ISO 5) vertical laminar flow air

• Polycarbonate and polypropylene design to

reflect UV energy

• Full access folding sash

• Unibody plastic design - no joints or gaps in

construction

• Digital UV light timer 0-59 minutes

• Lab event timer

Vibratome UltraPro 5000 Cryostat

The UltraPro 5000 open top cryostat has a comprehensive specification and offers the ultimate in

precision and constant reproducibility in the preparation of frozen sections. The 5000 cryostat has a 5040

rotary retracting microtome installed and is capable of sectioning a diverse range of hard and soft tissues

and many other materials. The advantage of utilizing cryo-sectioning in genomics research is that

chemical fixatives are generally not used in preparing tissues which promotes the preservation of antigenic

structures and macromolecules such as RNA and DNA.

Specifications:

• Minimum chamber temperature: -35° C

• Specimen temperature control; Fast reacting

from +5° C to minimum chamber temperature

• Quick Release Feather Blade Holder

• Specimen quick freezer running at -80° C

• Section range: 0.5 to 30 µm

Left: A chain of cells of the spiny diatom,

Chaetoceros sp., and a single cell of the diatom

Coscinodiscus sp. in the plankton from

Narragensett Bay, RI. [Differential interference

contrast transmitted light microscopy]

Right: LSM 700 confocal image

of the epithelium (EP) of the

mouse cecum showing bacteria

colonizing the mucus biofilm on

the surface. The bright orange

fluorescent bacteria are E. coli

cells that have been stained with

an RNA probe specific for this

species. The green cells were

stained with a non-specific dye

and represent all other bacteria in

this sample. Image courtesy of

Drs. Matt Mokszycki and Paul

Cohen, CMB, URI.