RESEARCHRESOURCESDIVISION

CBS Co-Creation of Knowledge Project

RRD

Research and Technology
SERVICES GUIDE- Published in 2024 -Research Resources Division
RIKEN Center for Brain Science
JULY, 2024

Unauthorized reproduction prohibited.

Contents
Research Resources Division (RRD) - Outline …………………………………………………………………… 1
Support Unit for Animal Resources Development

Facility for Mammalian Spacies ……………………………………………………………………………………………… 5
Staff-run Technical Services for Animal Resources Development ……………………………………………… 7
Mouse Embryo Manipulation …………………………………………………………………………………………… 7
Generation of Congenic Mice …………………………………………………………………………………………… 7
Generation of Transgenic Mice ………………………………………………………………………………………… 7
CRISPR/Cas9 Genome-Editing System ……………………………………………………………………………… 8
Generation of ES Cell-based Knockout Mice (Service in Suspension) ……………………………………8
ES Cell Establishment (Service in Suspension) ……………………………………………………………………8
Mouse Decontamination ……………………………………………………………………………………………………8
Rat Decontamination ……………………………………………………………………………………………………… 8
In Utero Electroporation (Service in Suspension) ……………………………………………………………… 8
Polyclonal Antibody Production ……………………………………………………………………………………… 8
Marmoset Supply …………………………………………………………………………………………………………… 8
Common Use Facilities and Materials ……………………………………………………………………………………… 9
Behavioral Testing Facilities for Mice and Rats ………………………………………………………………… 9Outline of Common Use Behavioral Testing Equipment ………………………………………………………10
Rental Rooms for Housing Special Animals ……………………………………………………………………… 11
Operation and Treatment Rooms for Monkeys (Macaques and Marmosets) ……………………… 11
Cooperative Use of Mouse Strains …………………………………………………………………………………… 11
Facility for Aquatic Animals …………………………………………………………………………………………………… 12

Support Unit for Bio-Material Analysis

Staff-run Technical Services for Bio-Material Analysis ………………………………………………………………13
DNA Sequencing (Plasmid Purification, Pretreatment, Sequencing) ……………………………………14
BioArray Analysis (Bioanalyzer) ……………………………………………………………………………………… 15
Genetic Polymorphism Analysis ……………………………………………………………………………………… 16
Gene Quantitative Analysis ……………………………………………………………………………………………… 17
BioArray Analysis (Gene-Chip Analysis) …………………………………………………………………………… 17
Next Generation Sequencing (Library Pretreatment, Sequencing) ………………………………………18
Mass Spectrometry ……………………………………………………………………………………………………………19
Peptide Synthesis …………………………………………………………………………………………………………… 21
Protein Purification ………………………………………………………………………………………………………… 22
Amino Acid Analysis …………………………………………………………………………………………………………22
Flow Cytometry and Cell Sorting ……………………………………………………………………………………… 23
Labware Washing …………………………………………………………………………………………………………… 23
List of Main Equipment for Staff-run Technical Services in the BMA ……………………………………24
Technical Personnel Support Section (TPSS) ……………………………………………………………………………25
TPSS: Molecular Biological Experiment …………………………………………………………………………… 26
TPSS: Histological Experiment (Histological Staining and Imaging) …………………………………… 26
TPSS: Histological Experiment (AAV Microinjection) …………………………………………………………27
TPSS: Bioimaging Experiment (Multi- Sectioning Image Acquisition using TissueCyte) ……… 27
TPSS: Virus Production Experiment ………………………………………………………………………………… 28
Shared Experiment Areas ……………………………………………………………………………………………………… 29
Common Use Equipment (CUE) Experimental Rooms …………………………………………………………29

Support Unit for Functional Magnetic Resonance Imaging ……………………………………………… 34
Support Unit for Electron Microscopy Techniques …………………………………………………………… 36
Appendix

Location of Common Facilities …………………………………………………………………………………………………37

Research Resources Division (RRD), RIKEN Center for Brain Science

Outline
The RRD is an organization that provides technical services anddevelopment primarily to members in the Center for Brain Science(CBS). Currently, the division consists of four support units asshown in the organizational chart on page 4, which cover a wide
range of technical fields.
Please refer to the website links below, and this guidebook fordetails concerning our versatile technical services.
https://cbs.riken.jp/en/faculty/rrd/

Grouping of RRD Services
The major services provided by RRD are categorized as follows:RRD Director
Hiroyuki Kamiguchi, M.D., Ph.D.

1. Staff-run technical services:
Following a request and samples from a research laboratory, knowledgeable andexperienced RRD technical staff perform analysis using optimal conditions or create tangible
objects based on samples.
The service contents are listed on the organizational chart on page 4. Most support can beapplied for online through the RIKEN Core Facilities Management System (R-COMS).
2. Technical personnel support section (TPSS):TPSS provides customized expert technical support for labs, such as helping out with lab
work when a lab is short-handed or providing technology that a lab itself does not have. TPSS
also provides support for the transfer of a lab’s newly developed technology to other labs.
3. Management services:
Shared experimental areas and common-use equipment are managed and maintained by the
RRD. Lab members can use them on their own by registering as a user and making an
advance reservation at R-COMS. A list of common-use equipment for each unit is shown on
pages 10.31-33.36.
4. Technical support activities:
RRD also organizes exhibitions of research materials and instructions of equipment to lab
members for educating scientific cutting-edge technology especially related to Neuroscience.
5. External cooperation :Collaborative research and commissioned tests will be used to disseminate some of the
RRD's research and technical support outside of RIKEN.

Users of RRD Services
The users of RRD services are primarily CBS members, as the RRD is part of CBS. However,as far as the situation of facilities, equipment and personnel permits, all RIKEN members
outside of CBS are eligible. In addition, non-RIKEN researchers also have a chance to usepart of staff-run technical services.

Service Fees
The expenses of the RRD are mainly covered by the CBS internal budget. Service fees arecharged for the use of RRD support services.

Proprietary Rights of Animals and Analyzed Data
When the RRD stores a lab’s experimental animals, samples, materials, or analyzed data aspart of its services, ownership of those materials belongs to the lab unless otherwise
agreed upon.
1

Publishing Scientific Data Acquired with RRD
So that we can continue providing technical support services, we ask that you credit RRD’s
specific contribution in the Materials and Methods and/or Acknowledgements section(s) ofyour paper when publishing research results obtained with our assistance. If the research
results were obtained via collaboration with our technical support, please indicate coauthorship in your publications.
Formal name of RRD
Center: RIKEN Center for Brain Science
Division: Research Resources Division
Units:
Support Unit for Animal Resources Development
Support Unit for Bio-Material Analysis
Support Unit for Functional Magnetic Resonance Imaging
Support Unit for Electron Microscopy Techniques
Acknowledgement Examples
Note: ###) Please enter the above formal name of support unit

1. Materials and Methods section (space dependent):*XXX was analyzed using the YYY service at the Support Unit for ### in RIKEN Center
for Brain Science, Research Resources Division (RRD).
2. Acknowledgments section (in each paper under three different cases):i) Without credit in the Materials and Methods section:*We are grateful to the Support Unit for ###, RIKEN CBS Research Resources Division,for technical help with XXX .
*We thank the Support Unit for ###, RIKEN CBS Research Resources Division,
with special thanks to Ms. YY and Mr. ZZ for XXX.
ii) With some credit in the Materials and Methods section:*We thank Ms. XXX for technical assistance with YYY.
3. With assistance from more than two RRD units:*We thank the staff of Research Resources Division, RIKEN Center for Brain Science,for generation of AAA mice and analysis of ZZZ.
4. Credit as a co-author:*Our affiliation is the Support Unit for ###, Research Resources Division, RIKEN Center
for Brain Science.

2

Number of RRD Staff(as of July 1, 2024)
Organization
Research Resources Division
Support Unit for Animal Resources Development
Animal Care and Management
Laboratory Animal Health Care
Staff-run Technical Services
Zebrafish Experiment Facility
OfficeSupport Unit for Bio-Material Analysis
Staff-run Technical Services& Shared Experimental Areas Management

No. of staff*(No. of part-timers)
1

Note

49
4
4(1)
15 (11)3 (1)contact employees

11 (1)
5
2(2)

TPSS

OfficeSupport Unit for Functional Magnetic Resonance Imaging
Staff-run Technical Services
OfficeSupport Unit for Electron Microscopy Techniques
Staff-run Technical Services
Office4121*Not included Director and Unit Leaders3Research Resources Division (RRD)
Director: Hiroyuki Kamiguchi, MD, PhD
Support Unit for Animal Resources Development (ARD)
Laboratory Animal
Facilities

Unit Leader: Kimie Niimi, DVM, PhD- Mammalian Animal Facility (mice, rats, rabbits, macaques, marmosets, and others)- Aquatic Animal Facility (zebrafish)

Mammalian
Staff-run Technical
Services

Complete Services:- Mouse Embryo Manipulation- Generation of Transgenic Mice
- Generation of Knockout Mice
- Mouse and Rat Decontamination- In Utero Electroporation- Marmoset Supply- Generation of Congenic Mice
- CRISPR/Cas System- Establishment of Embryonic Stem (ES) Cell
- Antibody Production- Animal health control- Marmoset Perfusion Fixation- Marmoset Surgical AssistanceTraining/Support:- Guidance and Support for Animal Experiments
Common Use
Facilities & Materials
Zebrafish
Staff-run Technical
Services

Common Use Facilities& Materials- Behavioral Testing Facilities for Mice and Rats
- Cooperative Use of Mouse Strains- Operation and Treatment Rooms for Macaques and Marmosets
Complete Services:- Zebrafish Embryo Manipulation - Generation of Transgenic Fish
- Generation of Knockout Fish
- CRISPR/Cas System- Sperm cryopreservation- Fish Health Care
- Housing and Breeding Facilities- Microinjector- Collection, Preservation and Provision for Zebrafish Strains

Support Unit for Bio-Material Analysis (BMA)

Unit Leader: Nobuhiko Miyasaka, PhD

Staff-run Technical
Services- DNA Sequencing (Plasmid Purification, Sequencing)- Gene Polymorphism Analysis- BioArray Analysis- FANTOM3 Clones Distribution- Amino Acid Analysis- Peptide Synthesis- Labware Washing- Genetic Quantitative Analysis- Next Generation Sequencing- Mass Spectrometry- Flow Cytometry and Cell Sorting- Protein purification- Lab Support Service

Technical Personnel
Support Section (TPSS)- Molecular Biological Experiments- Histological Experiments- Bioimaging Experiments- Virus Production Experiments

Shared Experimental
Areas (Self-run Use)

- Radiation Controlled Area, RI Center: CBS Common Use Facility(CBS Common Use Experimental Room)- Common Use Equipment (CUE) Experimental Rooms :・Molecular Biology Room・Gene Quantitative Analysis Room・Biochemistry Room・Chromatography Room
・P2/Level2 Experimental Room・Histological Preparation Room・Microscopy/Imaging Room #1, #2
・Flow Cytometry Room・Super Resolution Microscopy Room #1, #2- Freezer Room (Lending Service)

Technical Support
Activities- Research Equipment and Material Exhibition

External Cooperation- Commissioned Tests

Support Unit for Functional Magnetic Resonance Imaging (fMRI)- BMA Classes

Unit Leader: Tomohisa Okada, MD, PhD

Technical Services(Training/Support)- Human Subject Scans- Preliminary Experiments and Training- Monkey Subject Scans- Ethical Compliance Examinations

External Cooperation-Collaborative Research- Commissioned Tests

Support Unit for Electron Microscopy Techniques (EMT)

Unit Leader: Yoshiyuki Kubota, PhD

Technical Services(Training/Support)- Analysis of EM Data and Image Analysis for 3D Reconstruction- Analysis of Neuron Ultrastructure with High Pressure Freezing

Available Equipment- Focus Ion Beam-SEM- FE-SEM- Ultramicrotome - PC for Image Analysis

Center for Brain Science, Office of the Center Director
Services- Administrative Works for RRD - RRD system management - R-COMS support

4

July 1, 2024

Support Unit for Animal Resources Development
Support Unit for Animal Resources Development (ARD) maintains CBS’s well-equippedlaboratory animal-housing facilities and supplies reliable high-quality animals. The highlytrained technical staff provide services such as transgenic/knockout mice generation andmouse embryo manipulation. This unit also helps users to understand and follow official
procedures for animal experiments.

Facility for Mammalian Species
Animal Housing Capacity
The following table shows the total housing area and capacity for the housing of mammalian
species in each facility.
(As of JULY 1,2024)
CBS West Bldg.&
CBS East. Bldg.
Frontier Life
Science

CBS West Bldg.
CBS
CBS NCGR Bldg.
Annex
Ikenohata Bldg.

6,000m2(64,500ft2)

263m2(2,827ft2)

140m2(151ft2)

412m2(4,430ft2)

4,346m2(46,780ft2)

872m2(9,376ft2)

12,033m2(129,355ft2)

Housing capacity
Mice (5/cage)

6,000

50

-

-

14,000-20,050 (100,250 mice)Rats (3/cage)

-

-

-

-

840

-

840 (2,520 rats)

Rabbits (1/cage)

-

-

-

-

10

-

10

Macaques (1/cage)

-

20

29

5

-

-

54

Marmosets (4/cage)

-

-

-

59

-

-

59 (236 marmosets)

Marmosets (8/cage)

130

-

-

16

-

-

146 (1,168marmosets)

Building
Floor space

CBS Cent. Bldg.

Total

Animal Care and Management
The ARD facilities house SPF mice, rats, macaques, and marmosets, and conventional rabbits. In
principle, animals are owned by each research laboratory.
Animal care and management are carried out according to the standard operating procedure foreach animal species as defined by the ARD. The animal care technicians working in the facilities
are outsourced through professional companies that are contracted after a competitive bidding
process.

Animal Health Care
The A R D staff including four veterinarians, carries out the following operations on animal
health care.

Veterinary care in the animal facilities
Veterinarians perform the following:
Health monitoring of mice and rats; periodic health examinations of monkeys andmarmosets; diagnosis, treatment, and prevention of animal diseases; evaluation of healthstatus of incoming animals from outside institutions and vendors; quarantine of animals
procured; and certification of health status for animals exported to outside institutions.

Arrangement, maintenance and management of animal facilities
Veterinarians oversee animal care and management, arrange facilities, coordinate use of
facility space, and approve animal facility visitors.

5Microbiological monitoring procedures
The microbiological monitoring procedures for maintaining the health of mice/rats
conducted by the ARD are described below; the test result information may be provided tooutside institutions when mice/rats are shipped from ARD facilities.
Mice:
Three to seven monitoring cages each housing 1 to 3 sentinel mice are placed in eachanimal room. During cage exchanges (done every two weeks), bedding taken from several
rearing cages in the same room is placed in the monitoring cage. Sentinel mice which havebeen in the monitoring for at least 3 months are used for microbiological testing.
Rats:
Three to six monitoring cages each housing 1 to 3 sentinel rats are placed in each animalroom. During cage exchanges (done every week), bedding taken from several rearing
cages in the same room is placed in the monitoring cage. Sentinel rats which have been in
the monitoring cage for at least 3 months are used for microbiological testing.
Testing of sentinel mice/rats:
Internal testing is performed by A R D veterinarians and staff once every 3months. External testing is outsourced to the Central Institute for Experimental
Animals (International Council for Laboratory Animal Science Monitoring Center) once
every 6 months.
The average number of sentinel mice and rats in each animal room is indicated below.
Internal testingMice: 2 mice each are selected from 2 monitoring cages which have been
kept for at least 3 months in each animal room.
Rats: 1 rat each is selected from 2 monitoring cages which havebeen
kept for at least 3 months in each animal room.
External testing:

1 mouse or rat which has been raised in a monitoring cage for at least 6months is randomly selected and sent out for external testing.

Evaluation of test results
The test results are analyzed by the veterinarian in charge and compiled in a
microbiological test result chart. When issues arise based on the test results, instructions
on countermeasures are given to staff and users.

6

Staff-run Technical Services for Animal ResourcesDevelopment
Technicians in the Support Unit for Animal Resources Development provide the following
services for researchers.

Mouse Embryo Manipulation
In vitro Fertilization (IVF)
Oocytes collected from superovulated females are fertilized with matured sperm collectedfrom males. The embryos after in vitro fertilization can be cryo-preserved or transferred topseudo-pregnant females to obtain pups.

Micro-insemination (Intracytoplasmic sperm injection: ICSI)
Immature sperms collected from infant or infertile males are injected into the cytoplasm ofoocytes under a microscope to obtain embryos. Sperms with impaired motility collected fromsterile males are also available.

Embryo Transfer
Embryos are transferred into the oviduct of recipients. This method can be used to obtaindecontaminated mice and a large number of age-matched mice.

Fertilized Egg Freezing and Cryopreservation
Valuable mouse strains are maintained in CBS for future neuroscience research. Fertilized
eggs taken from these mice are frozen by vitrification methods and preserved in liquid
nitrogen to maximize resources.

Sperm Freezing and Cryopreservation
Valuable mouse strains are maintained in CBS for future neuroscience research. Sperm takenfrom these mice are frozen and preserved in liquid nitrogen.

Reanimation of Mice from Cryopreserved Fertilized Eggs
Cryopreserved fertilized eggs from ARD stocks or introduced from outside institutions arethawed and used for embryo transfer as described above.

Reanimation of Mice from Cryopreserved Sperms
Frozen sperms from ARD stocks or introduced from outside institutions are thawed and used
for in vitro fertilization. Fertilized eggs are then used for embryo transfer as described above.

Generation of Congenic Mice
When you wish to replace the genetic background of genetically modified mice with a B6/J
strain or other strain, in vitro fertilization can accelerate in producing the next generation ofoffspring compared with the natural mating. We are able to perform backcrossing for thedesired number of generations.

Generation of Transgenic Mice
Plasmids are purified with a QIAGEN plasmid kit or equivalent, digested with restriction
enzymes (to remove the backbone vector), run on an agarose gel, and a gel fragment containing
the transgene is excised. The transgene is purified using the QIAGEN QIAquick Gel Extraction kit.
The researchers adjusts the transgene to a concentration of 20~50ng/μl and submits 50μl (inthe case of BAC transgene, 50μl of 1-10ng/μl solution) for microinjection into fertilized mouse
eggs. The injected eggs are transferred at the 2-cell stage into the oviduct of recipient females.
Tail samples from a few weeks-old mice are provided to the researchers for genotyping, andcreated transgenic founder mice are provided to the researchers. It takes approximately twomonths to obtain transgenic founder mice.
7

CRISPR/Cas9 Genome-Editing System
The guide RNA complementary to the target sequence and Cas9 mRNA (or protein) are prepared by
the researchers. They are microinjected into 250 fertilized eggs, and then the injected eggs aretransferred at the 2-cell stage into the oviduct of recipient females. Tail samples from 2-week- old miceare provided to the researchers for genotyping, and created genome-edited founder mice areprovided to the researchers. It takes approximately two months to obtain genome- edited foundermice. This service was started from FY2014.

Generation of ES Cell-based Knockout Mice (Service in Suspension)
Targeting vectors constructed by researchers are introduced into embryonic stem (ES) cells
using electroporation and approximately 300 ES cell colonies are picked up. Positive clones
selected by the researchers are injected into blastocysts. The injected blastocysts are transferred
into recipient mice. The researchers receive the chimera mice and examine them for germline
transmission. It takes approximately three to four months to obtain chimeramice.

ES Cell Establishment (Service in Suspension)
Embryonic stem (ES) cells can be established from genetically modified mice generated bylaboratories. It takes approximately three weeks to establish at least two lines of ES cells (forexample mice with a C57BL/6 genetic background).

Mouse Decontamination
Decontamination by embryo transfer
Embryos are transferred into the oviduct of SPF recipients.

Decontamination by hysterectomy
Fetuses at gestational days 18 and 19 are removed from the mother’s uterus using sterile
techniques and reared by a SPF foster mother.

Rat Decontamination
Decontamination by hysterectomy
Fetuses at gestational days 21 and 22 are removed from the mother’s uterus using sterile
techniques and reared by a SPF foster mother.

In Utero Electroporation (Service in Suspension)
In utero electroporation is a useful method for transfecting neuronal progenitor cells in vivo. By
adjusting the placement position of the electrodes and the developmental time-point ofelectroporation, specific subsets of neurons can betargeted.

Polyclonal Antibody Production
To produce polyclonal antibodies, researchers provide immunogens to the staff foradministration to the desired animal(s): rabbits, mice, or rats. The antibody titers are checkedwith ELISA tests.

Marmoset Supply
Marmoset breeding started in 2007 to provide marmosets to laboratories in CBS. Currently, thefacility is maintaining 25 pairs capable of breeding approximately 50 offspring per year.

8Common Use Facilities and Materials

Behavioral Testing Facilities for Mice and Rats
The behavioral testing facilities for mice and rats are grouped into two categories as follows.
A. Behavioral testing rooms for common use
These are common-use rooms for all CBS laboratories. The A R D manages the room andmaintains the equipment.
Locations: Facilities are located on the 8th floor of the CBS Cent Bldg. and the 2nd and 3rd floorof the CBS NCGR Bldg.
Reservations: Reservations to use the rooms can be made through the R-COMS system.
Rooms can be reserved for fixed hours or days. The maximum period that rooms
can be reserved is 15 days, not includingholidays.
Long-term use: The term of use is decided between individual laboratories and the ARD.
User fees: The fees are charged per hour or day of use based on the reservation record by the
R-COMS system.
Special experiment area: In this area, the researchers can infect mice with viral vectors(P1A/P2A level) and perform behavioral testing of the infected mice.
B. Rental rooms
The ARD lends the rooms to laboratories for a fixed term (ex. 3 months, 6 months or 1 year).
Experimental equipment is to be supplied, managed, and maintained by the individuallaboratories.
Locations: Facilities are located on the 2nd floor and 3rd floors of the CBS NCGR Bldg.
Use: The term of use is decided between individual laboratories and the ARD.
User fees: CBS extra lab room charge facility use expenses.

9Outline of Common Use Behavioral Testing Equipment
Current
Equipment ID
Building

CBS Central

Current Room No.

(Use this ID
when booking)Former Room
Original
No. ( CBS West
Equipment ID
Bldg. )

Behavior Testing Equipment & Facilities

Sound
attenuatingroom

N810-1

WC0535

107-1

WC0535Mice long-term behavioral rhythms analysis (30ch)

Yes

N810-2

WC0536

107-3

WC0536Morris water mazeNone

N810-3WC0543205

WC0543

Contextual and cued fear conditioning test (2ch)

None

206

WC0960

Passive avoidance test (8ch)

None

205-1

WC0961

Barnes circular maze

None

-

-

Eight-arm radical maze

None

-

-

Dissection room

None

205-A

WC05533-chambers analysis

None

205-C

WC0959Open field test 600x600 (4ch)

Yes

N810-4

WC1176

N810-5

WC0539N810-6

WC0959N811-1

WC1177

205-D

WC0965Light/dark transition test (4ch)

Yes

N811-2

WC0544205

WC0964

Startle response & Pre-pulse inhibitation test (4ch)

None

205-B

WC0966

Tail suspension & Porsolt forced swim test (2ch)

None

205-F

WC0545

Elevated plus maze (double)

Yes

205-F

WC0545Y-mazeYes

205-B

WC0966Ultrasonic analysis

None

206

WC0960Rotarod test

None

206

WC0960Hole-board test

None

205-E

WC0548Open field test 500x500 (4ch)

Yes

206

WC0960Contextual and cued fear conditioning test (2ch)

None

-

-

(Treatment room)

None

107-4

WC0551Forced swimming test

None

107-4

WC0551

Metabolic cage

None

205-1

WC0961Five choice serial reaction time test

None

N811-3A
WC0545
N811-3B

N811-4

WC0547

N811-5

WC0548N811-6

WC0550

N812

WC0551

N814

WC0552

107-1

WC0552

Scanet 12ch

Yes

N815

WC0554--(Treatment room)

None

301c

WC0528Startle response & Pre-pulse inhibitation test (4ch)

None

301c

WC0541

Rotarod test（1 ch）x 2 sets

None

301c-1

WC0526

Tail suspension & Porsolt forced swim test (2ch)Yes301c-2

WC0531Morris water mazeYes

301c-3

WC0532

Contextual and cued fear conditioning test (2ch)Yes

Elevated plus maze (single)
311c-1

WC0530311c-2

WC0527

Light/dark transition test (4ch)Open field test 500x500 (4ch)

CBS NCGR

Activity Sensor system

Yes
Yes

311e

WC0529202a-

Rental room

None

202c

-

Rental room

None

202d

-

Rental room

None

WC1016

Intellicage ( 4 ch )None

WC1017

Intellicage ( 4 ch )None

WC1018

Five choice serial reaction time test（ 12 ch ）

None

202f

-

Rental room

None

202h

-

Rental room

None

202e

Scanet10

YesRental Rooms for Housing Special Animals
Housing rooms for special animals are equipped in room 506 the CBS East Bldg. These rooms areavailable for rent by laboratories for housing special animals (e.g., ferrets, prairie voles) andmice/rats. Users are responsible for all animal care, including installing housing equipment. Users
can move animals freely between these rooms and their laboratories.

Operation and Treatment Rooms for Monkeys (Macaques and Marmosets)
The ARD is equipped with an operating room for cranial and other surgery, a procedure/autopsy
room for formalin fixation, etc., an ICU room for post-operative care or care for animals in poor
condition, and a treatment room for drug administration, etc. These rooms can be used by alllaboratories, and are managed by ARD.
Locations:
The facilities are located in rooms 310 and 311 of the CBS West Bldg., N905-1/S906-1/
S906-2/S906-3 of the CBS Central Bldg.
Reservations:
Reservations to use these rooms can be made through the R-COMS system from the
COMMON website.
User fees:
Fees are charged by time or day of use based on the reservation record by the R-COMS
system.

Cooperative Use of Mouse Strains
CBS researchers can cooperatively use JAX Mice purchased from the Jackson Laboratory by CBS
labs. The list of JAX Mice is available on the web. http://cbs.intra.riken.jp/mouse/11Facility for Aquatic Animals
Zebrafish Experiment Facility
This facility was set up at the CBS Ikenohata Bldg. in March 2004. The breeding area is 872m2
and the laboratory area is 365m2. The breeding area has the following kinds of tanks: 40
outsized (extra-large), 4,320 large-scale, 2,160 medium and 4,120 small, with a total breeding
capability of about 200,000 zebrafish. A high-quality control of water purity is maintained inthis facility, and the water temperature is kept at 28.0~28.5℃. The laboratory areaaccommodates apparatus and materials for embryonic manipulation, microscopy, and strain
storage.
This facility is also a core facility of the National BioResource Project Zebrafish (Dr. Okamoto,
Senior Visiting Scientist of CBS). The core facility’s goal is to collect, preserve, and provide
zebrafish strains as a useful vertebrate model in Japan.
From April 2018, we started the charging system for these support services.

Technical Support Services
The Zebrafish Experiment Facility provides the following support and training services for users.

Zebrafish Maintenance and Breeding
Fish maintenance in the facility is carried out by RRD staff according to the CBS Zebrafish
Facility Manual. When using the Aquatic Animal Experiment Facility for the first time, theusers must submit an application form and attend facility orientation sessions. A separate
application is required to use zebrafish rearing facilities.

Zebrafish Sperm Cryopreservation
Zebrafish sperm is frozen to preserve strains. We recommend using 3-5 male zebrafish perstrain.

Generation of Transgenic/Knockout/Knockin Zebrafish
We offer many services, from construction of plasmids for injection to manufacturingtransgenic fish, transgenic fish sorting and strain maintenance.
Plasmid Construction: Plasmids for producing transgenic fish are constructed based on
the vector containing Tol2 transposon elements. Constructed
vectors for transgenic lines with modified bacterial artificial
chromosomes (BACs) or synthesis guide RNA/Cas9 mRNA forknock-out, knock-in, etc. also available.
Injection: Plasmids, modified bacterial artificial chromosomes (BACs) or
guide RNA/Cas9 mRNA are injected into fertilized eggs; embryos
are bred for about 4-5 months in the breeding room and thenprovided to users.
Screening and Maintenance: After crossing, founder fish are selected by genomic PCR analysisof the DNA from the embryo or by observation of fluorescentmarker proteins in embryos using a fluorescent microscope.
Guided by the staff, users perform screenings and maintenance ofthe fish.

Support for Equipment Use
Equipment (Behavioral experiments, electrophysiology, imaging, etc.) in the facility
is available for users with support and training by the staff.

12Support Unit for Bio-Material Analysis
The Support Unit for Bio-Material Analysis (BMA) provides the Staff-run Technical Services, including
bio-material analyses (e.g., nucleic acids, proteins, and amino acids), cell sorting, and peptide synthesis.
In addition, customized technical services have been offered by expert technicians of the Technicalpersonnel Support Section (TPSS) since December 2018. The unit also maintains and supervises the
Shared Experimental Areas. These areas include the Common Use Equipment (CUE) Experimental
Rooms, where both general-purpose and highly technical equipment are available for shared use invarious research projects. Additionally, the BMA holds ‘BMA Class’ and ‘RRD Exhibition for ResearchMaterials’ for effective utilization of staff-run technical services and CUE in CBS.
BMA internal website: http://common.riken.jp/rrd/ComInstrue/index.html

Staff-run Technical Services for Bio-Material Analysis
For Users
・Procedure
Users (possessing RIKEN ID) wishing to order technical services should first read the guidelines
for desired services on the above BMA website and confirm the equipment to be used, content of
measurement/analysis, and fees. The experiment plan should also be discussed with the person in
charge as necessary.
・Operating hours
RIKEN normal working hours are observed. If an operation must be temporarily shut down formaintenance or other official events, an announcement will be made. These can be found on the“News & Announcements” section of the BMA internal website.
・Management of analyzed data
Analyzed original data will be kept on file at RRD for a period of five years, except the data fromhuman materials of some services (e.g., DNA Sequencing). We will also discard all data when a lab
is closed.
・Publishing Scientific Data Acquired with RRD (Also see page 2)
We would like to ask users to credit our specific contribution when publishing research resultsthat were obtained with the assistance of our staff-run technical services. Please see more details
in page 2 of this service guide.

13

DNA Sequencing (Plasmid Purification, Pretreatment, Sequencing)
CBS Cent. Bldg., N010/011

1. Support Services
Plasmid Purification
Transformed bacteria with plasmid DNA is received from the user and thenincubated in the medium overnight. Plasmids are extracted by alkaline-SDS
and silica membrane methods using a Biomek® i7 automated workstation(Beckman Coulter). The purification system is set up in either a 96-wellformat or an 8-well format.
The 384-well plate Pipetting service is transferred from a 384 well-plate to
4 x 96 well-plate.

FX Robotic workstation(Beckman Coulter)

Pretreatment for Sequencing (Cycle Sequencing Reaction)
Cycle sequencing is performed using purified plasmids or PCR products as templates. After cycle
sequencing, the samples are automatically transferred to the DNA Sequencing Service. The service also
accepts GC rich samples and samples that require refined purification of the PCR product (ExoSAP
treatment).
DNA Sequencing (Sanger Method)
Redundant terminators in the sample are removed using the CleanSEQ Kit(Beckman Coulter) magnetic beads. The DNA sequencing analysis isconducted with the 3730xl DNA analyzer (Applied Biosystems).

Users are able to order from Plasmid Purification to DNA Sequencing
simultaneously.

3730xl DNA Analyzer(Applied Biosystems)

2. Outline (Methods and Results)
Protocol for Plasmid DNA Purification① Incubate pre-cultured media with bacterial cells at 37℃ for 17 hr.
② Harvest bacterial cells③ Resuspend bacterial cells with Resuspension Buffer④ Lyse bacterial cells by adding Lysis Buffer and mixing⑤ Neutralize by adding Neutralization Buffer and mixing⑥ Transfer crude lysates onto the filter and clear crude lysates by
centrifuge⑦ Transfer cleared crude lysates onto the DNA binding filter andbind DNA to silica membrane of the filter by centrifuge⑧ After washing the silica membrane by Wash Buffer, elute plasmid
DNA by Elution Buffer

Yield of purified plasmid DNA

①

②

③

④

20

10

10

0

⑧

HEK293 cells

30

20

⑦

The fluorescent-expressing plasmid pmCyan-N1 ispurified from E. coli strain DH5α using Nucleospin
Multi-96 Kit.

40

Recover DNA (mg)

Recover D N A (m g )30⑥

Transfection of cell lines

抽出プ ラ スミ ド の収量
40

⑤

0

50

5 0Elution volume (µL) 1 1 0
Elu tion Volu m e (µL)
110

The right figure shows the transfection efficiency
with HEK293 cells using.
Lipofectamine 2000 (Invitrogen).
Transfection of HeLa and N2a cells is also available.

14

Microscopic Image

Pretreatment for Sequencing[Cycle Sequencing Reaction][Reaction Solution]
Sample (Template DNA + Primer)

5.0 µlBigDye Terminator v3.1

1.0 µl

5 x Sequencing Buffer

3.5 µlDeionized Distilled Water

10.5 µl

Total

20.0 µl

DNA Sequencing[Cycle Sequencing Product Purification][Result of Electrophoresis]

Redundant terminators are removed using
CleanSEQ Kit magnetic beads.

ACGCCGGACTTGTCC
CTTGCGCGGAACGTAGAGGAG~

SampleMagnetElectropherogram（ABI3730xl）

CleanSEQ
Analysis Data (Software: 4 Peaks)

BioArray Analysis (Bioanalyzer)
CBS Cent. Bldg., S004

1. Support Services
Total RNA degradation and DNA fragment size are checked by the Agilent 2100 Bioanalyzer.
This equipment is also used for quality check (QC) of the next generation sequencing (NGS) samples.

2. Outline (Methods and Results)
Sample concentration
RNA Nano Chip: 25 – 500 ng/μL
RNA Pico Chip: 200 – 5000 pg/μL
DNA 1000 Chip: 0.1 – 50 ng/μL
High Sensivity DNA Chip: 5 – 500 pg/μL
The RIN is calculated automaticallyby the Bioanalyzer for 18S, 28S rRNA.
(RIN: RNA Integrity Number, Index of the RNA quality)
A RIN of more than 7 is usually used for experimentation. (Max10)15

Genetic Polymorphism Analysis
CBS Cent. Bldg., N010/011

1. Support Services
Fragment Analysis *Fluorescence-labeled DNA fragments are subject to capillary electrophoresis using the 3130xl Genetic
Analyzer (Applied Biosystems), to detect the size or mobility.
Genotyping① Genomic DNA Extraction
Mouse or rat genomic DNA is purified from tails or ears using the Agencourt DNAdvance Kit(Beckman Coulter). The genotyping service is also available for other samples ( tails of zebrafish,
nails of humans and so on) .
② PCR *
Fluorescence-labeled DNA fragments are amplified using fluorescence-labeled primer(s).
③ Analysis of peak size *Labeled DNA fragments are electrophoresed using the 3130xl Genetic Analyzer.

④qPCR genotyping
Genotyping using the real-time PCR device for genomic DNA (①) extracted from the biologicalsample.

* When conditions for the analysis are not decided, we examine the optimum. (additional charge)
2. Outline (Methods and Results)
Fragment Analysis
SampleElectrophoresis conditions of 3130xl

Sample Preparation(Denaturation)
96℃ 2 min → On ice

Change parameters based on:
Fluorescence labeled PCR product
Size standard Labeling kit

Genotyping
Sample< e.g. >Mouse Tail 2 - 3 mm
Mouse Ear 1 - 2 punch

Electrophoresis
using 3130xl
Gene A
Gene B
Gene C① Genomic DNA Extraction② PCR③ Analysis of peak size

Treatment of samples

PCR

Electrophoresis
using 3130xl

Template DNA 10 ng
Primer 0.2 µM eachTaq 0.25 U

Genome DNA
DNAdvance<Examples>
WT

Gene I
Exon A

KO①

Exon B
Exon C②
About 100 bp

Exon D

Wild type

Gene II

Exon A①

Knockout③
About 200 bp① Labeled Fwd primer (WT, KO)② Non-labeled Rev primer (WT)
③ Non-labeled Rev primer (KO)Heterozygous

Genotyping - qPCR Sample< e.g. >＜例＞ マウス
Mouse Tail2 - 3 mm
Mouse Ear 1 - 2 punch①Genomic DNA Extraction④qPCR genotyping

Treatment of samples

qPCR

Genome DNA
DNAdvance

16

Template DNA 50 ng
Primer 0.3 μM each
THNDERBIRD 10μl

Example of result
QuantStudio12K

Gene Quantitative Analysis
CBS Cent. Bldg., C011

1. Support Services
Gene Expression Quantification
TaqMan Assay or SYBR Green Assay: The sample, primer, probe (for TaqMan assay only) and enzyme
are mixed and then monitored for fluorescent intensity using the Real-Time PCR machine(QuantStudio 12K or 7900HT, Applied Biosystems).
Genotyping For SNP
TaqMan Assay: The sample, primer, two TaqMan probes and enzyme are mixed. The SNP signal isdetected using the Real-Time PCR machine.
Mouse Genotyping
TaqMan Assay or SYBR Green Assay: The sample, primer, probe (for TaqMan assay only) and enzyme
are mixed and then monitored for fluorescent intensity using the Real-Time PCR machine.
Nonspecific Reaction Check
User-designed primers can be checked for nonspecific reaction activity.
QuantStudio 12K(Applied Biosystems)

2. Outline (Methods)
PCR Reaction (Default)
TaqMan Assay

PCR Reaction
40 Cycles
95℃

95℃

10 min 15 sec

95℃

95℃

15 sec15 secMix
SYBR Green Assay
50℃

60℃

60℃

1 min

15 sec2 min
UDG
Activation
Incubation

Denature

Anneal＆ExtendDissociation Stage (SYBR Green Assay)

3. Outline (Results)
Amplification Plot

Results Table

For each sample:
the quantity is calculated using the Ct value of the standard curve.
The Ct is set to be within the
exponential growth region of theamplification plot. The threshold isthe line whose intersection with theamplification plot defines the Ct.

ExampleStandard Curve Plot

The standard curve plots the Ct values
vs. the quantity of the standard samples.

1.Relative RNA value (X) is normalized among GAPDH values(Internal Control).
2.Result of Probe A (Y) was normalized with the X value.
3. A relative value for Probe A is calculated as the ratio between
two sample's Y/X values.

17

Next Generation Sequencing (Library Pretreatment, Sequencing)
CBS Cent. Bldg., S004

1. Support Services

2. Procedure

Library Preparation
・Genomic DNA enrichment including exons using
SureSelect and library preparation for nextgeneration sequencing (NGS).

Cooperative support
Sequencing
through outsourcing

Sequencing
at RRD

Pre-Meeting (BMA)• Other support applications:e.g. Whole Genome, ChIP-Seq, Methyl-Seq,
RNA-Seq, Single-cell RNA Seq [Quartz-Seq2].

Specifications

Sequencing using MiSeq• Deep sequencing Using MiSeq is performed in BMA.
• Cluster generation, and Sequencing by the MiSeq(Illumina). FASTQ files are generated from thesequence data.

Purchase Library Prep Kit (by User)

Prepare Sample(DNA, RNA etc.)

3. Outline (Methods and Results)
Submit Samples

Example: Exon analysis using SureSelect• Genome DNA (Using 3ug)

To Cooperative
Supports• Shearing using Covaris (150bp)

To NGS Service• Add adapter• Hybridize sample with biotinylated cRNA
Library (Exon)

Meet/Consult• Collect sample using streptavidin and enrich• Add index

Order Service• Check size & Quantitative analysis

Purchase Sequence
Reagents (User)

Focusedultrasonicator(Covaris)• Sequence

・Sequencing at the BMA

Desktop Next
Generation Sequencer(MiSeq)Submit Samples

Receive Data(FASTQ File)• Sample QC

・Sequencing through Outsourcing• Cluster generation

At the consultation, decide on a kit to use, etc.
2. BMA coordinates library preparation . The
service corresponds to the following
applications; Exome, RNA-Seq, ChIP-Seq,
Methyl-Seq
3. Quality Checks are conducted by the
Bioanalyzer and MiSeq
4. KAPA kit is used for the quantitative analysis
5. Sequencing at the cooperative outsource.
1.

• Sequencing (*24 hours)• FASTQ Generation• Receive Data

*150bp Paired Run (v2 Kit)

ResultCluster image

Quality Score

>=Q30
44.4G
92.4%
FASTQ image*Researchers analyze data on the FASTQ files
using 3rd party software.

18

Mass Spectrometry
1. Support Services

CBS Cent. Bldg., S002

2. Procedure

Molecular Weight Determination
Electro-spray ionization (ESI)-mass spectrometer (MS) or
Matrix-assisted laser desorption ionization (MALDI)-MS areused to determine molecular weights of organic compounds.
High resolution and high accuracy measurements are alsoavailable using the ESI-MS.
Protein Identification (ID) and Post-Translational
Modification Analysis
The protease-digested peptides are analyzed using
the LC-ESI-MS/MS. For high accuracy identification andmodification analysis, digested peptides are analyzed using
the quadrupole-orbitrap MS. Proteome analysis for relative
protein quantitation are also available by stable isotope
labeling methods among multiple samples and/or label freemethod (LFQ).
Protein / Small Molecule Quantitation
Target proteins or small molecules are quantified by MRM(Multiple Reaction Monitoring) method using the triplequadrupole MS with a suitable LC. For absolute quantitation,
addition of stable isotope labeled internal standards isrecommended.

Molecular
weightDetermination

Protein ID, Quantitation,
Small molecule
Post-translational
Quantitation
modification analysis

Meet/Consult(Receive
Digestion kit)

MS and HPLC
method optimization

Digest Protein

Sample clean up
optimization

Submit Samples〜1 day〜2 weeks〜1 week

Receive Data

3. Outline (Methods and Results)
Molecular Weight Determination
MALDI

Select Matrix

ESI

Calibration

Calibration

Sample/Matrix Co-crystallization on a Target Plate

Measurement(Desalt)
Sample Loading Nanospray Tip

Measurement

Analysis (BioPharma Finder)

QExactive
autoflex

・ Detection sensitivity: fmol-pmol range.
・ Do not use detergent. If it is necessary, limit the detergent to a minimal amount. Samples may require ZipTip
processing to concentrate and/or desalt prior to analysis.

Protein ID & Post-Translational Modification Analysis
Prepare Sample(Filtrate or Desalt)

Determine LC/MS
Conditions

LC-MS/MS
Analysis

DB Search(MASCOT and/or
Proteome Discoverer)

Modification Analysis
ManualComparison of Specific and Ubiquitous Protein Expressions among Mouse Brain Regions,Cerebral Cortex (CTX), Hippocampus (HIP), and Cerebellum (CER)
LC-MS/MS Analysis

Protein Identification

Total of 4587 proteins were identified from
triplicate samples of the three brain regions.

Vanquish Neo + QExactive
・ We recommend performing all protein digestions at the clean bench (N005).
・ Taxonomy information is required for a MASCOT database search.

19Comprehensive and Relative Protein Quantitation Analysis
Determine LC/MS
Conditions

Desalt-Stable isotope labeling method-

Data Analysis(Proteome Discoverer)-Label-free method (LFQ)Comparison of Protein Expression
between CTX and CER
Volcano Plots

MS/MS spectrum (TMT labeling)*

DB Search(MASCOT and
Proteome Discoverer)

LC-MS/MS
Analysis

Sequence Information
Quantitative Information*

127N 127C

126 127N 128N 129N 130N 131N
127C 128C 129C 130C 131C

Vanquish Neo + QExactive

・ Stable isotope labeling methods (e.g. iTRAQ/TMT, SILAC, and 18O) and label-free method
are available for comprehensive and relative protein quantitation among multiple samples.

Targeted Protein and Small Molecule Quantitation Analysis--Multiple Reaction Monitoring (MRM) methodMake thePrimary
Transition List

MRM
Analysis(1)

Determine LC/MS
Conditions

Methionine cycle metabolites
Cystathione
CholineBetaineIntensity

Intensity

Native peptide

Isotope-labeled peptide(Internal Standard)

SAM
Methionine
SAH

x
1/10

0
Retention time (min)Data Analysis(Pinpoint orTraceFinder)

Vanquish UHPLC + TSQ Altis- Quantitation using MRM method Peptide (angiotensin-II)*

MRM
Analysis(2)

Fix theTransition List

1

2
3
Retention time (min)

5MTHF
x 100

4

Easy-nLC 1200 + TSQ Vantage
・ MRM is an analytical method with high sensitivity; it selectively detects only target peptides or small molecules(Maximum transitions: 3,000 MRMs per run).
・ Proteins with MRM transitions list created by the RRD are able to be analyzed without method development.
・ As a first step for quantitation of new compounds, optimization of various protocol conditions such as sample clean-up steps (byuser) and analytical methods for UHPLC and TSQ Vantage (by BMA staff) are required.
* Modified from user’s publication: Mol Cell. 2015 Jun 18;58(6):1015-27.

20

Peptide Synthesis
CBS Cent. Bldg., S003

1. Support Services
Peptide synthesis:
Peptides are synthesized by the solid phase method using automated peptide synthesizers; quality isdetermined by HPLC and MALDI-TOF/MS analysis. Crude peptides or purified peptides in lyophilized
form are provided to users.
Modifications:
Conjugation to a carrier protein (KLH or BSA) for antibody production.
N-terminal : Free, Ac, Fmoc
C-terminal : Free, NH2, Resin
Special synthesis:
Long chain peptides (≧31aa), 250μmol scale, biotinylation, cyclization, fluorescent label, peptide
synthesis including unusual amino acids and phosphoamino acids.

2. Outline (Methods and Results)
Solid-phase peptide synthesis

Synthesis
Peptides are synthesized by the solid phase method; using a peptide synthesizer, they are built "backwards"
from the C-terminal to the N-terminal.
Three different peptide synthesizers are used, suitable for each synthesis scale and sequence.

433A

Liberty Blue

Purification of peptides

MultiPep CF & MicroColumn

The target peptide is separated and collected by HPLC system. Quality of the crude and purified target peptides isdetermined by HPLC and MALDI-TOF/MS analysis.
Crude peptide
Crude peptide
HPLC

TargetMS

TargetPurified peptide

Chromastermicroflex

Purif ied peptide
HPLC

HPLC conditions:
Column, Inertsil ODS-3 (250 x 4.6 mm I.D.)
Column temp., 25℃
Mobile phase, 1-51% CH3CN (contg. 0.1% TFA) Analytical time, 50 min
Flow rate, 1.0 mL/min
Detection, UV at
215nm

21

MS

TargetProtein Purification
CBS Cent. Bldg., C013

2. Outline (Methods and Results)

1. Support Services
・Antibody Purification
Antibodies are purified from plasma andsera by AKTA (Cytiva).

Antibody Purification Flow

Affinity Chromatogram
mAU
5000

Affinity column
Preparation4000~ 1 day・Protein Purification[Biological Proteins]
Particular proteins are purified from biomaterial, such as brain tissue, and cells.
Products and data (e.g. chromatograms)
are returned to users.
User-designed purification protocols arewelcome.

3000

Affinity
purification

2000

1000

Gel Filtration

0

Gel Filtration
Product andData Return

mAU
5000

2 days ~ 1 week

[Target Proteins]
Purification of the following proteins areavailable: Recombinant proteins, His-tag
fusion proteins, GST fusion proteins, IgG
antibodies, and serine proteases.

AKTA Explorer 10

4000

3000

2000

1000

0

Purified by the selected purification method(determined at the preliminary meeting/consultation).

Amino Acid Analysis
CBS Cent. Bldg., C013

2. Outline (Methods and Results)

1. Support Services

Ninhydrin method- Amino Acid Composition Analysis
The quantitative analysis of free amino acids, amino
sugar and protein hydrolysate is performed by anamino acid analyzer using the post-column method.
Fluorescent method– Neurotransmitter Amino Acid Analysis
The quantitative analysis of amino acids (glutamate,aspartate, and GABA) in brain tissue and brain
microdialysates is accomplished on a high-performanceliquid chromatograph (HPLC) with fluorescentdetectors (FLD) using the pre-column method.

Neurotransmitter

Monoamine

DL-Amino Acid
Electrochemical
methodFluorescent method(OPA method)
Analysis

1day 〜 1 week

Return Data

Fluorescent method -Neurotransmitter Amino Acid Analysis

Neuromodulator Analysis
The quantitative analysis of a non-listed small
molecule as the above technical service isperformed using HPLC with FLD/UV/ECD detector
・Development of analytical method for a small
molecule to chose a detector and examining the
optimization of the analysis condition.
・The quantitative analysis by HPLC
(FLD/UV/ECD)
22

5-HT

3-MT

HVA

ISO

DA
5-HIAA

NA

NM

MHPG

DOPAC

Electrochemical method -Monoamine Analysis

EpiElectrochemical method– Monoamine Analysis
The quantitative analysis of monoamines(noradrenaline, dopamine, serotonin) and its
metabolites in the brain tissue extracts orbrain microdialysates are performed using
HPLC with an electrochemical detector (ECD).

Receive Sample

Flow Cytometry and Cell Sorting
CBS Cent. Bldg., S008

1. Support Services•

Cells specifically labeled with fluorescent dyes or expressing
fluorescent proteins are separated using a fluorescence-activated cell
sorter, FACSAria (Becton Dickinson).

•Able to perform the index sorting that is useful for one cell analysis.

•

Isolated cell preparations, together with the property diagram
analyzed by software FlowJo (Becton Dickinson) for flow cytometry(FCM) analysis and the microscopic data obtained under an Olympus
Fluorescence Microscope (EVIDENCE), are returned to researchers.

2. Outline (Methods and Results)
Example: Pretreatment

384 well plate
single cell sorting

Sasagawa et al. Genome Biology 19:299, 2018

Example: Results
An example of flow cytometry analysis and cell sortingwith YFP expressing cells from mouse brain (Cited from reference)

Extract tissue from brain*if possible, remove the white matterCut tissue to 1-2 mm then transfer to 50 mL
Incubate the papain solution containing
the tissue at 37℃ with agitation
Triturate with pipette
2 times
Filtrate through 40 μm nylon mesh
Triturate with pipette tip
Filtrate through 40 μm nylon mesh
Add propidium iodide and Hoechst33342
Flow Cytometry analysis and cell sorting

The target cells are detected by using several parameters.
(A) High level of Hoechst 33342 detected nuclei (in the polygonal gate),(B) Doublets or multiplets and debris excluded using FSC/SSC dot plots (outside of thepolygonal gate), (C) Histogram plot indicating selected live cells. Dead cell populations are
localized in the high PI fluorescent range, (D) FSC and YFP two parameter dot plots indicate
the sorted cell population (in the quadrangle gate), (E) Frozen tissue section of the cortex, (F)Dissected cortex, (G and H) Triturated cell mixtures before cell sorting, (I-K) YFP expressing
cells purified by cell sorting. Scale bars: 200μm in E, 20μm in F-K .
Reference; Ohtawa and Mataga, SEITAI NO KAGAKU 62(2): 165 - 170, 2011.

Labware Washing
CBS Cent. Bldg., S001

1. Support Services
・CBS Central Bldg.
Labware washing, drying, and sterilization (by heat treatment or autoclave).
・All CBS Bldgs.
Pipet tip packing and sterilization (by autoclave).

2. Outline (Methods and Results)
Order: Bring labware with completed application form to the RRD washing room.

Washing: Using a pH neutral non-phosphate liquid detergent (2% Scat 20X-N) in an ultrasoniccleaning machine.
Rinse: Using tap and pure water in an automatic washing machine.
Drying: The washed labware is dried, and then capped with aluminum foil.
Heat Treatment: Please let us know if this step is necessary for your experimental purpose.
23

List of Main Equipment for Staff-run Technical Services in the BMA(as of July 1, 2024)
Equipment

Manufacturer

Model

Location

DNA Sequencing
DNA Sequencer

Applied Biosystems

3730xlCBS Cent. N011

Liquid Handling Workstation

Beckman Coulter

Biomek i7

CBS Cent. N010

Applied Biosystems

7900HT, QuantStudio 12K

CBS Cent. C011

Applied Biosystems

3130xlCBS Cent. N011

Agilent

Agilent 2100 Bioanalyzer

CBS Cent. S004

illumina

MiSeq

CBS Cent. C011

Bruker Daltonics

autoflex

CBS Cent. S002

Gene Quantitative Analysis
Real-Time PCR System

Genetic Polymorphism Analysis
Genetic Analyzer

BioArray Analysis
Bioanalyzer

Next Generation Sequencing
Next Generation Sequencer

Mass Spectrometry
MALDI TOF/TOF Mass Spectrometer
Triple Quadrupole Mass Spectrometer
Quadrupole-Orbitrap Mass Spectrometer
Nanoflow HPLCTSQ Altis/Vantage
Thermo Fisher Scientific

Ultra Performance Liquid chromatography

Q Exactive
EASY nLC1200/Vanquish Neo

CBS Cent. S002/N008

Vanquish UHPLC

Peptide Synthesis
Thermo Fisher Scientific
Peptide Synthesizer

433A Peptide Synthesizer
Liberty Blue

CEM Corporation

MultiPep CF
CBS Cent. S003

Hitachi High-Technologies

L-7450 and others

GL Sciences

PU714M, BG-34-02, UV702
and others

MALDI TOF Mass Spectrometer

Bruker Daltonics

microflex

AKTA Protein Purification System

CytivaAKTA explorer10S with Frac-950
AKTA prime

Hitachi High-Technologies

L-8900

Shimadzu

LC-10Avp

Eicom

700 Series Sytem

Eicom

HITEC-500

CBS Cent. N004

Becton Dickinson

FACS Aria II SORP, FACSAria SORP,
FACSymphony A3

CBS Cent. S008

HPLC

CBS Cent. C013

Amino Acid Analysis
Amino Acid Analyzer

HPLC

CBS Cent. C013

Flow Cytometry and Cell Sorting
Flow Cytometry System

24Technical Personnel Support Section (TPSS)
The BMA set up a new section for providing customized support services termed Technical
Personnel Support Section (TPSS) in October 2018 and allocated expert technicians. Purpose of
the services is to help labs closely with technical support to meet their requirements. For example,
TPSS staff perform routine experiments in the lab according to their experimental protocol. As
advanced services, techniques not available in the lab are also provided by expert TPSS staff to do
experiments and/or operate state-of-the-art instruments. Furthermore, we actively try to receive
technical transfer of a newly developed technique from a lab and then provide it to different labs as
an advanced service by TPSS.
http://common.riken.jp/rrd/cominstrue/subaEng/tpss/tpss.html

For Users
・Procedure:
1. At the beginning to use desired service, users should discuss with the person in charge in BMA
about experimental plan including service contents and schedule. The BMA unit leader willattend the meeting as necessary.
2. Application can be done through R-COMS. Application forms for uploading the R-COMS can beobtained from the website above.
・Operating hours:
RIKEN normal working hours are observed and advanced reservation is always necessary.
・Management of analyzed data:
Analyzed original data will be kept on file at RRD for a period of five years, except the data fromhuman materials of some services (e.g., Validation of data ). We will also discard all data when
the corresponding lab is closed.
・Publishing Scientific Data Acquired with RRD
Please credit our specific contribution in the methods and/or acknowledgments section(s) of yourpaper (please see page 3 for details). As a special notes, when a TPSS staff made a significant
contribution to your research, we would appreciate it if you could acknowledge the person or
include the person as a co-author of your paper.

Features of Services by TPSS staff:
Molecular Biological Experiment
Plasmid construction and amplification
Histological Experiment
AAV Microinjection under Stereotaxic Conditions
Histological Staining and Imaging• Perfusion Fixation• Immunohistochemistry• In situ hybridization• LacZ, Nissl, CO, and Hematoxylin (HE) staining• Embedding• Imaging by digital scanners/microscopes
Bioimaging ExperimentMulti- Sectioning Image Acquisition using TissueCyte
Virus Production Experiment
Production of AAV vector, lenti virus vector, rabies virus vector e.t.c.

25

TPSS: Molecular Biological Experiment
CBS Cent. Bldg., C010
1. Support Contents► Plasmid Construction
This support constructs new plasmids. The original plasmid DNA provided by the user are
recombined and reconstructed using the method requested by the user. Consultations on
design are also available.
► Plasmid Amplification
The required amount of plasmid DNA is amplified and purified from E. coli.
A QIAGEN kit is used for purification. The yield estimates for each column size are as follows:
Miniprep: up to 20 μg Midiprep: up to 100 μg Maxiprep: up to 500 μg

2. Outline (Method and Results)
Recombine the insert gene and vector provided by the user using the recombination
method requested by the user↓
Select clones using the method requested by the user↓
Perform sequence analysis as necessary(Request BMA technical support DNA sequence analysis)↓
Amplify and purify to the requested yield↓
Prepare glycerol stock if requested↓
Measure DNA concentration↓
Hand over the final product and report

TPSS: Histological Experiment (Histological Staining and Imaging)
CBS Cent. Bldg., C005

1. Support Contents• Services from histological preparation using mouse brain tissue to its imaging are provided.
• You are able to order a part of work contents, 1. to 5. below.
1. Perfusion fixation
2. Tissue sectioning (freezing sample, non-freezing sample)
3. Staining by order methods(e.g. Immunostaining, In situ Hybridization, Nissl, Hematoxylin/Eosin staining)
4. Mounting tissue on slide glasses
5. Image acquisition (By using RRD CUE digital slide scanner )2. Outline (Method and Results)<View of Sectioning><Procedure>
Using users’ materials (e.g. samples, chemicals) and theirexperimental protocols, customized support services are
performed. Rats and zebra fish are also acceptable to theservice with a prior consultation held.
＜Staining example＞

Freezing Microtome
YAMATOREM-700

Nissl Staining

26

HE Staining

TPSS: Histological Experiment (AAV Microinjection)
CBS Cent. Bldg., N815 , Neural Circuit Genetics Bldg., 311f

1. Support Contents

2. Outline (Method and Results)＜Procedure＞• Leica Angle Two Dual Stereotaxic/
Mouse is used to perform AAV
injection in the mouse brain.

Pull a glass capillary (Sutter, Micropipette puller P-97）↓

Attach the glass capillary to one side of a Teflon tube
and set it on the Angle Two arm.
Attach the opposite side of the Teflon tube
to the Hamilton syringe and set it to the syringe pump.

• Other viral vectors, retrograde oranterograde tracers, chemicals arealso available to injection.

↓

Fix anesthetized mouse to stereotaxic apparatus.
↓

Incise the mouse head to expose the skull.
↓Start up the Angle Two system.
↓

Adjust the height of the brain under the microscope.
↓

Check the coordinate of the target and make a hole in the skull.
↓

Suction up AAV from the tip of capillary and align to the target.
↓

Inject AAV↓

Pull out the capillary from the brain and suture the skin.
↓

Leica Angle Two Dual Stereotaxic/ Mouse

Stop anesthesia, remove the mouse from the stereotaxic
and return it to a cage.

TPSS: Bioimaging Experiment (Multi- Sectioning Image Acquisition using TissueCyte)
CBS Cent., C005, C107

1. Support Contents

2. Outline (Method and Results)＜Procedure＞

Multi- Sectioning Image Acquisition using TissueCyte

TissueCyte 1400FC (TissueVision) is a device that can
image each cross-section as a wide-range fluorescence
image with a two-photon laser microscope whileautomatically sectioning the brain of a small animal suchas mice and zebrafish in the XY direction with a highperformance vibratome.
This support includes from embedding of mouse brain
tissue to stitching of 2D tiling images.

Preparation of brain block (embedding)Set the brain block to the TissueCyte
Setting of various conditions for imaging
Imaging

Ragan, T., et al. (2012). Serial
two-photon tomography for
automated ex vivo mousebrain imaging. Nat. Methods 9,

Image processing (stitching)

255–258.

Return data and sample
TissueCyte 1400FC＜Example： B6.Cg-Tg(Thy1-YFP)HJrs/J ＞
TissueCyte Stitching software27

3D construction (Image J )TPSS: Virus Production Experiment
CBS Cent. Bldg., C010

・ Adeno-associated virus (AAV)
１． Support Contents

2． Procedure• Provide custom-made AAV vectors (P1 level)• Respond technical consultation with users
concerning AAV production

In the safety cabinet, experiments are done.
Preparation culture cell and plasmids↓
Transfection of plasmids & AAV production↓
Harvest AAV from supernatant & cells↓
Concentration of supernatant by PEG↓
Ultracentrifuge (purification/concentration)↓
Titer check（qPCR or dPCR)

The followings are discussed with users at a premeeting, and we will produce the ordered AAV.
a. AAV’s serotype
b. Expressing genec. Titer (gc/mL) & volume (µL)
◆ Please prepare Transfer-plasmid by users.
◆ In some cases, we would like to ask users to

prepare serotype-plasmid.

・ Rabies virus (RV)
１． Support Contents

2． Procedure• Provide custom-made RV vectors (P2 level)• Respond technical consultation with users
concerning RV production

In the safety cabinet, experiments are done.

For custom-made RV production, we confirm user’s
status (whether the user obtain the followings).
A. Plasmids and cells for the RV production
B. Approval for RV experiment

Preparation culture cell and plasmids↓
Transfection of plasmids & RV production↓
Harvest RV & RV amplification↓
Exchange glycoprotein of RV (eg. EnvA)↓
Ultracentrifuge (purification/concentration)↓
Titer check

The followings are to be discussed with the user at
the pre-meeting, and we will produce the ordered RV.
a. Strain and envelope proteinb. Expressing genec. Titer (IU/mL) and volume (µL)
※Please prepare all cells and plasmids for RV by users.

・ Lentivirus (LV)
１． Support Contents

2． Procedure• Provide custom-made LV vectors (P2 level)• Respond technical consultation with users
concerning LV production

In the safety cabinet, experiments are done.
Preparation culture cell and plasmids↓
Transfection of plasmids & LV production↓
Harvest LV from supernatant↓Ultracentrifuge (purification/concentration)↓
Titer check（qPCR or dPCR)

The followings are discussed with users at a premeeting, and we will produce the ordered LV.
a. LV’s envelope proteinb. Expressing genec. Titer (gc/mL) & volume (µL)
※ Please prepare all plasmids for LV by users.
28

Shared Experiment Areas
BMA manages and maintains the Shared Experimental Areas. The Common Use Equipment (CUE)
Experimental Rooms are located in the CBS Cent. Bldg. (B1F, 1F). CBS Common Use Facility for unsealed
radioisotope experiments is located in the RI Center. These experimental areas are available for use ofboth CBS and all other labs in RIKEN.

Common Use Equipment (CUE) Experimental Rooms
http://common.riken.jp/rrd/cominstrue/subaEng/indexe/indexcome.html

1. Support Services
Widely use and/or highly technical equipment are organized into groups and installed in separate
rooms in the CBS Cent. Bldg. according to the purpose of use (B1F: 10 rooms, 1F: 1 room) .
Upon completion of user registration online, the equipment and working spaces in these rooms areavailable for use (self-run user).

2. Procedure
・Users
A user is any person in RIKEN given permission to access the Experimental Rooms by the Unit Leaderof the Support Unit for Bio-Material Analysis (hereinafter referred to as "UL").
・Applicant
1) To use the CUE Experimental Rooms in the CBS Cent. Bldg., registration is necessary (The RIKEN
Core Facility Management System, R-COMS) will use for the registration from April 1, 2023).
2) Orientation (only for new users) and System Key Box registration (according to the approved
application) will take place by a CUE Administrative Officer
3) To conduct experiment that requires careful attention to safety management; All experiment plansincluding the CUE Experimental Room numbers need to be approved/issued by the Safety Division
before initiating the experiments.
・User registration validity
Users are permitted to use the facility from the date their application is approved until one day
before of approval of annual registration renewal for the following year.
The renewal notice will be sent from BMA to budget managers and users at the early fiscal year.
・Conditions of use
1. The user can use all equipment in the Experimental Rooms after UL approval. . However, if users
wish to conduct an experiment that requires careful safety management, they must haveapproved or issued receipt for all registered experiments by the Safety Division before initiating
experiments.
2. CUE must be operated by the user. Be aware that these are shared use equipment; read instruction
manuals that accompany the equipment and follow instructions. Certain designated equipment
requires special instruction for beginners and/or users by the CUE Equipment Adviser or
manufacturer. For more information, please contact the adviser.
3. The annual registration user fees are required for the use of the CUE Experimental Rooms. An
additional fee is charged for specific equipment requiring special consumables and/or annual
contracts for maintenance. Concerning other equipment with free usage fees, consumables such as
reagents will be borne by the user’s Lab.
4. Workspaces for sample preparation are organized for each experimental room. In-use reagents andsolvents can be temporarily stored in the rooms but return all waste to the user's lab (exceptions:liquid waste for chromatograph analysis, autoclaved culture-liquid waste, etc.).
5. Contact the CUE Equipment Adviser promptly if there are any problems with the equipment. In caseof any damage to the equipment, whether or not it is the fault of the user, the adviser should be
notified immediately. All repairs must be made following the adviser's instructions. If the user isclearly at fault for equipment damage, the user's lab is responsible for repair costs.
・Hours of use
There are no restrictions. The key to each laboratory on the B1F is a key box, and the key to eachlaboratory on the 1F is controlled by the IC-key.

29

・Equipment reservation
Shared facilities/equipment reservations are available online. Use the R-COMS (RIKEN Core Facility
Management System) to make advance reservations (reservations and changes can also be made on
the shared PC in the laboratory).
https://riken.simprent.jp/users/login?locale=en
・Logbooks
Enter the required items (name, lab and etc) in the logbook attached to the piece of equipment.
Note: CUE equipment that has not been used/reserved more than 1 year may be discontinued.

3. Features of the CUE Experimental Rooms and Main Equipment Lists(As of April 1, 2024)

・Molecular Biology Room (N002)
This room contains basic equipment for molecular biology such as thermal cyclers andspectrophotometers. The Live Cell imaging and Analysis System and Gene transfection System areinstalled. Genetic recombination experiment is also available under P1, P1A (only C.elegans) and P2
in this room.
・Gene Quantitative Analysis Room (C011)

The Real-Time PCR systems and desktop next generation sequencer are used for staff-run analysis
and self-run use.
・P2/Level 2 Experimental Room (S009)
Sample preparation space is available under P1, P2, Level 1 and Level 2. This room is also available
for shared use to maintain cultured cells, including microorganisms such as viruses (Level 1, Level 2),
and Living Modified Organisms (P1, P2).
・Histological Preparation Room (C004)
This room contains, all processing equipment needed for histological preparation and observation(from tissue fixation embedding, and sectioning to staining). It is also equipped with cryostats forfreezing specimens.
・Microscopy/Imaging Room #1 (N003)
General-purpose all-in-one fluorescence microscopes and slide scanners are installed in this room.
There are also imaging systems for chemiluminescence, fluorescence, and colorimetric imaging
applications.
・Microscopy/Imaging Room #2 (C107, 1F)
High magnification confocal laser scanning microscopes are installed in this room under atemperature-controlled environment. Neurolucida system is also available in this room.
・Super-Resolution Microscope Room #1 (N005-1)
The room contains a confocal laser microscope with super resolution, time gate, high speed scan, andhigh-sensitive photon counting functions.
・Super-Resolution Microscope Room #2 (C012-1)
The room contains a spinning disk confocal super resolution microscope for high-speed imaging
including a live cell imaging and Lattice Structured Illumination Microscopy System with lattice SIM
system, a CO2 incubator and a clean bench.
・Chromatography Room (N004)
Several chromatography systems for analysis of proteins, peptides, and neurotransmitters are
installed in this room. Among these, HPLC conditions have been established for the following
methods: simultaneous analysis of monoamines and their metabolites, high-sensitive dopamine &serotonin analysis, and simultaneous analysis of glutamate & GABA.
・Biochemistry Room (N005)
Basic equipment for biochemistry, such as microplate readers, a centrifugal evaporator and freeze
dryer are available in this room. One of the key features of this room is the installation of several
pieces of special equipment, such as Biacore and NanoSight.
30

・Flow Cytometry Room (S008)
The Flow Cytometer, the flow cytometry software “Flowjo” and Automated cell isolation system
which is available for the pretreatment of FCM’s samples are installed in this room; other pieces ofequipment are used for Staff-run Technical Service.
・ Other available equipment
Besides the CUE, BMA also manages and maintains the CBS Common Use Equipment, Mass
Spectrometry Room (C308-2) and equipment for microdialysis sampling.

<< All common Use Equipment are available to use by members of CBS Co-Creation Labs >>

31

32

33

Support Unit for Functional Magnetic Resonance Imaging
Equipped with a 3-Tesla MRI system and a 7-Tesla MRI system, we support non-invasive
functional MRI (fMRI) studies on human subjects and animals conducted by laboratories
within RIKEN. Besides fMRI studies which are the primary focus of our MRI facility, we also
have the capability to perform DTI, MRA, MRS, and simultaneous EEG/fMRI measurements.
We have installed a number of peripheral devices for fMRI studies, such as stimulus
presentation devices (video projectors, earphones, an electrical stimulation device, etc.), tomeet researchers’ various needs for experiments. Subject condition (heartbeat, respiration,eye movement, pupil size, EMG, button press response, etc.), stimulus timing, and the shape of
gradient pulses can be recorded together with the acquisition trigger timing signal, enabling
this information to be used for post-processing and analysis of the MRI data. We have unique,in-house developed pulse sequences, data analyzing processes and peripheral equipment.
Multi-shot EPI for high spatial resolution imaging, software for regressing out noise due toboth respiration and cardiac pulsation, and a bite-bar system for minimizing subject motion
are all available for users. We have put special emphasis on developing software to process kspace (raw) data so that we are not limited by the vendor’s processing stream, which is
optimized for clinical applications rather than research. We have developed software forreconstruction, noise reduction, and visualization of data in variety of formats. Using thesecutting-edge technologies, users can perform many types of experiments and optimallyprocess the data according to their needs. For researchers outside RIKEN, MRI experiments
can be performed as Commissioned Test.
MRI spec
Items

3T MRI

7T MRIMAGNETOM Prisma

SIGNA™ 7.0T

Siemens

GE

Static field(Tesla)

2.89

7

Max Gradient(mT/m)

80

100

Max slew rate(T/m/s)

200

200

Number of RF reception

64

64

Number of RF transmission

2

8

Body RF coil

Yes

No

Helium consumption(L/month)00March 2015

April 2023

Product name
VenderDate of introduction

34Peripheral devices
Projector WUX5000 (Canon)
LCD Goggle device (NNL)
Audio stimulation device
Physiological signal sensors（Heartbeat, Respiration, etc.）• EEG device available under MRI environment• Response button devices• Optical fiber thermometer

•
•
•
•

•
•
•
•

Eyeglasses for MRI
Bite-bar system for head stabilization
Computer for stimulus device control
Computer for analog signal recording

Collaborative Research and Commissioned Test
There are two means available to users outside RIKEN. Either they can conduct measurements as part ofan internal RIKEN experimental program after signing a joint research agreement with a RIKEN
laboratory, or they can conduct measurements using the commissioned exam system. In the latter case,only data acquisition is provided. In the case of commissioned exams on human subjects, in particular, itis necessary to undergo ethical review outside RIKEN. In addition, if you wish to use experimental
equipment that is not already available, a safety review will be required.
Collaborative Research Procedures
After discussing the experiment with the RIKEN laboratories, including our unit, and obtaining theagreement of all parties, a joint research plan will be established at both facilities. If the measurement
involves human subjects, the research will be subject to RIKEN's internal ethical review. It is important
to clarify the payment of fees as this may not be available depending on the type of budget.
Procedure for Using Commissioned TestFirst, please consult with our unit. Depending on the measurement details, this service may not be
available. If the project is deemed acceptable, you will be asked to apply for contract testing. Afterpaying the fee according to the plan, the experiment will be started. After all the planned experiments
are completed, the contract will be terminated when the measurement data is delivered to you.

35

Support Unit for Electron Microscopy Techniques
The Support Unit for Electron Microscopy Techniques (EMT) provides facilities and technical support
for research that requires ultrastructural context, such as synaptic connections, and fine structuresof intracellular organelles. The staff has specialized knowledge and skills for EM, and works withindividual research laboratories to understand their specific needs, proposes options for researchmethods to achieve their research goals, and provide the best technical support possible at the Support
Unit. We also host seminars to train individual researchers and trainees on various EM technologies
available at the Support Unit.

Technical Services Available for Electron Microscopy Techniques
The Support Unit for Electron Microscopy Techniques provides the following technical support with indepth training on the use of these microscopes to assist researchers to obtain high-quality images required
for their research.

Electron Microscopy
Technical Services Provided by the Staff
Preparation of tissue for electron microscopy:
Biological specimens such as brains and organs are chemically fixed, stained with metal, and embedded inresin. The tissue can also be processed with a high-pressure freezing, resin replacement, and embedding for
better preservation of its ultrastructure. Before the experiment, we would like to have a good discussion
with researchers to understand their research project to select the best tissue processing method for the
research project.
Electron microscope:a) FIB-SEM (Focused Ion Beam-Scanning Electron Microscope)
The block surface revealed by thin (>4 nm) milling by the FIB is imaged using SEM. Repeating this process
will produce serial electron micrographs automatically. The effective milling depth is about 100 μm;
therefore, we recommend an imaging area of about 100 x 100 μm2. With this technique, the data obtained
are in digital form, while physical tissue block portion are lost.
b) FE-SEM (Field Emission-Scanning Electron Microscope)
Serial ultra-thin sections obtained with an ultramicrotome are collected on either wafer or tape, and theregion of interest is imaged using the FE-SEM. Relatively larger image areas are possible. Each section canbe re-viewed multiple times for higher power or different regions of interest. Section thickness is >40nm
thick.
Analysis of the EM data sets:
Instructions are provided on image analysis tools for 3D reconstruction, including NIH-image, VAST-Lite,and Amira.

Available Equipment
The Support Unit for Electron Microscopy Techniques provides the following EM equipment on B1F and 1Fof the Brain Science Central Building.
(List of main equipment)
Equipment

Manufacturer

Model

FIB-SEM

ThermoFisher

Helios 5

FE-SEM

JEOL

IT-800Ultramicrotome

Leica Microsystems

ARTOS-3D

ATUM

RMC

ATUM

Ultramicrotome

Leica Microsystems

UC-6, FC-6

High pressure freeze eq.

Leica Microsystems

EM ICE

Opto/electrical stimulation Unit

Leica Microsystems

36

Appendix

Location of Common Facilities

CBS NCGR Bldg. 3F :Animal facility [mice / rats / rabbits / manipulation room / behavior testing rooms]

Vivarium Area (P2A)
P2A対応動物実験施設

CBS NCGR Bldg. 2F :Animal facility [mice / rats / behavior testing rooms / cage washing room]37

CBS Cent. Bldg. 8F : Animal facility [mice / mouse behavior analysis rooms / quarantine room]

CBS Cent. Bldg. 9F : Animal facility [marmosets]

38

WC

附属実験施設

102

１階平面図
101
階段
Frontier Life Science Research Facilities 2F:
Animal facility [marmosets]

West Building
225

228

224

（226）

207

208

Research Resources Division
206

Storage

227 229

205

207-1
暗室2

204-1

Shower209
Storage

room

204-2

EV

204

222

鏡検室
203

EV

221-1

機械室
202-2

Marmoset
212

RRD
213

RRD
211

RRD
214

RRD
210

202-1

WC WC
202
倉庫

RRD
215

201
階段

Frontier Life Science Research Facilities

バルコニー
312
マーモセット312-1 飼育室
CBS West Bldg. Annex : Animal
facility [macaques, marmosets]
飼育室1
-2

312

308

307

306

マーモセット 廊下
飼育室2

脳統合機能T（谷藤TL)

305
研究基盤開発部門
304-4

マーモセット
飼育室3
EV

311
-3

-1
-2

310

303-2

309
機械室

EV

310-1

EV

303
303-1

非常階段

西棟
WC WC

附属実験施設

脳統合機能

302
倉庫

３階平面図

301
階段

39

302
-1

302-2

CBS East Bldg. 5F : Animal facility [marmosets / special animals]

CBS East Bldg. B1F :CBS West Bldg. 3F :Animal facility [macaques]

40

Animal facility [macaques], MRI room

CBS Ikenohata Bldg. 2F : Zebrafish facility

RI Center : RI facility

41

CBS Cent. Bldg. B1F :Bio-material analysis room [staff-run/TPSS/ common use equipment (CUE)],
Electron microscopy room, Cryopreservation room

Machinery

Machinery

Okano Laboratory
fMRI
MRI Room

Dry PitOperation
Room

B03

B08

B09

B09-1

B10

B04
MachineryRRD Laboratory
(CUE)

B01

RRD
Lab.
(CUE)

TanakaLab.

Freezer
Room

RRD
Laboratory

RRD
Laboratory(EMT)

N005-1

N006

N007

N008

N009

RRD
Laboratory(TPSS)

RRD
Laboratory
(CUE)

C010

C011

RRD Laboratory(CUE)

MachineryRRD Laboratory

Storage

WC

N001

N002

N003

N004

N005

EV
hall

N011

C007

RRD
Lab.
(EMT)

RRD
Laboratory
(CUE)

RRD
Laboratory(TPSS)

RRD
Laboratory
(EMT)

C003

C004

C005

C006

EV

WC
EV

C009

C008

RRD Lab.
(CUE)
C012-1Low
Temperature Room

RRD
Laboratory

StairsEV
hall

Storage

Stairs
C002

Storage

C001

Storage

EV

C103

C012

C015

C014

WC

WC

RRD
Laboratory

RRD Laboratory

RRD
Laboratory

Large
FormatPrinter

RRD Lab.
Meeting
Room

RRD
Storage

S005
-1

S005
-2

Machinery
B02

S001

S002

S003

S004

S005

S009

RRD Office

Machinery
S006

S007

S008

RRD Lab.
(CUE/TPSS)

Dry Pit

C000 Cryopreservation

Stairs

N010

B12

CBS Cent. Bldg. 1F:
Bio-material analysis room [common use equipment (CUE)], Electron microscopy room,
Office of the Center Director

Dry PitUR

UR
Office of the Center Director

Brain Box

N102

CEV
EV

N105

N106

N108

Conference
Room

RRD Lab.
(CUE)

C106

C107-1

C107-2

StorageC101-1

Seminar Room

SafetyControl

StairsC104

C105

EV Hall

DS

RRD
C101

EV

C102

C103

C1051

CBS
Mail-box Room
C108

C109-1RRD Exhibition
Room
RRD Lab. (EMT)
C109

EV
Hall

Stairs

EV
C110

S107

Receiving Room
S101-1

Storage
C108

S105-2

S101-2

PDFA

S101
Masao Ito lounge

Library

TULLY'S
COFFEE

UR
S102(S103)

S104

S105

42

S106

Storage

Garbage

N107

WC

N104

WC

WC
Stairs

N103

WC

S108
Neuroinformatics
Unit

S109
BrainMINDS
OfficeUR

Dry Pit

N101

StorageRRD Office(EMT)

Entrance Hall

WC

EV
Hall

CPR
Meeting
Room