Repertoire Genesis, Inc.
Technical Guide

For sample processing and shipping
Ver.2016.03.31

Table of contents
1: Introduction ································································ 1
2: Processing and shipping method ···································· 1
3: PBMCs, cultured cells or sorted cells ······························· 2
5: Tissue ········································································· 3※Please contact us if you have any questions about this material
Repertoire Genesis, Inc.
104 Saito Bioincubator, 7-7-15 Saito-Asagi
Ibaraki-shi, Osaka, 567-0085 Japan
TEL: +81-72-657-8686, FAX: +81-72-657-8687
E-mail: info@repertoire.co.jp

For sample processing and shipping
Repertoire Genesis, Inc. Ver.2016.03.31
1: Introduction
This material provides the protocol for processing the specimens and sending thesamples to us for performing the TCR/BCR repertoire analysis. We conduct therepertoire analysis based on total RNA, which tends to be degraded easily. Whentotal RNA is degraded, it is not possible to obtain a good result. Therefore, we
conduct a quality check using Agilent 2200 TapeStation for all samples. Please be
sure to read this document before you initiate sample processing. In this technicalguide, we will introduce a stabilization example using RNAprotect cell Reagent orRNAlater and a shipping method.

2: Processing and shipping method
In our repertoire analysis, we use specimens that include the mRNA of T cells and B cells (blood,cultured cells, sorted cells and tissue).
The processing and delivery methods are shown in the following table. More details will be
described separately as per the type of specimen, later in this document.

SampleBlood

Cultured cells

Type(PBMC isolation)sorted cells

Processing

Shipping

RNAprotect cell reagent

Shipping with refrigerant packTissueRNA*

RNAlater
Shipping withdry ice*We do not recommend you to send us RNA, because dry ice might be vaporized duringtransportation and delivery is sometimes delayed. If you want to send us RNA stored or freshly
extracted in your lab, please call us.

― 1 ―For sample processing and shipping
Repertoire Genesis, Inc. Ver.2016.03.31
3: PBMCs, cultured cells or sorted cells
After you isolate PBMCs from whole blood using density gradient centrifugation (e.g. FicollPaque™, Lymphoprep™), you can stabilize RNA in cells and store the cells with RNAprotect cell
reagent for up to 7days at 15-20℃ or for up to 4 weeks at 2-8℃. For culture cells and sortedcells, you can recover the cells by centrifugation and perform stabilization of cells with theRNAprotect cell reagent.

Reagents
RNAprotect® Cell Reagent (cat#: 76526, QIAGEN)

Stabilization protocolCells in solution

1.

Wash cells (<1 × 107 cells) with medium or buffer (e.g, RPMI1640, Hanks, PBS)

2.

Re-suspend cells with 1 vol. of medium or buffer3.

Add 5 vol. of RNAprotect cell reagent

4.

Mix cells by shaking, pipetting, or vortexing

Cell pellet

1.

Cell pellets (<1 × 107 cells)

2.

300 μL of RNAprotect cell reagent (>5 vol. of cell pellet)

3.

Re-suspend the cells completely by vortexing.

Shipping protocol
1.

Placed 1.5- or 2-mL sample tubes into a box with a lid

2.

Put the sample box into the Styrofoam box according to our Packaging Instruction

3.

Include a "shipping specimen list"

4.

Wrap the package with strong tape (cloth duct tape)

5.

Ship the package to address below by FEDEX

6.

E-mail us the sample list and notify the tracking number

Shipping address:

Repertoire Genesis, Inc.
104 Saito Bioincubator, 7-7-15 Saito-Asagi
Ibaraki-shi, Osaka, 567-0085 JapanTEL: +81-72-657-8686, E-mail: info@repertoire.co.jp― 2 ―For sample processing and shipping
Repertoire Genesis, Inc. Ver.2016.03.31
4: Tissue
In case of fresh tissues, you can stabilize RNA by immersion of the tissues with RNAlater andstore the immersed tissues for up to 7days at 15-20℃ or for up to 4 weeks at 2-8℃.

Reagents
RNAlater® Stabilization Solution (Cat#: AM7024, Ambion)

Stabilization protocol
1.

Dispense 1 mL of RNAlater into 1.5-mL tubes inadvance.

2.

Trim the tissue specimens to a weight of 100 mg(approximately half the size of a soybean).

3.

Cut into 3~5-mm3 sections and immerse them inRNAlater.

4.

After immersion, store the samples as soon as possible
in the refrigerator (4℃) (overnight to 4 weeks).

5.

For long-term storage, remove the RNAlater reagent
after overnight immersion, and store the samples at −80℃ (optional)

Shipping protocol
1.

Placed 1.5- or 2-mL sample tubes into a box with a lid

2.

Put the sample box into the Styrofoam box according to our Packaging Instruction

3.

Include a "shipping specimen list"

4.

Wrap the package with strong tape (cloth duct tape)

5.

Ship the package to address below by FEDEX

6.

E-mail us the sample list and notify the tracking number

Shipping address:

Repertoire Genesis, Inc.
104 Saito Bioincubator, 7-7-15 Saito-Asagi
Ibaraki-shi, Osaka, 567-0085 Japan
TEL: +81-72-657-8686, E-mail: info@repertoire.co.jp― 3 ―Packing Instruction
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Freeze ice packs overnight
Seal sample tubes (1.5 mL Eppendorf tube)
and place them in a sample box.
Use a Styrofoam box with walls 2 inches thickor morePlace a ice pack in the bottom of box
Place a thin layer of 3-4 paper towels (forinsulation)
Put the sample box on itFill the box with necessary ice packs
Fill all gaps along box and between ice packand box with paper towel
Seal the box with Sealing Tape
Ship the box by the FEDEXPaper towel
Sample box
Thick-wall Styrofoam ice box(Ex. 15" x 15" x 12")Required ice pack: 10 kg (3-days)

Ice pack