Tissue Sample Submission Guideline - BGI A0 Tissue Sample... · Blood Preserve in Paxgene Blood RNA...

Tissue Sample Submission Guideline

Document NO.: SOP-SMM-033

Version NO.: A1

Effective Date: 2018-1-3

Document NO.：SOP-SMM-033 Version NO.：A1 Page 1 of 21

Directory

ABOUT THIS GUIDELINE .............................................................................................................................................. 3

NOTES AND PRECAUTIONS: ........................................................................................................................................ 4

1 SAMPLE REQUIREMENTS .................................................................................................................................... 6

1.1 REQUIREMENTS FOR DNA EXTRACTION ............................................................................................................... 6

1.1.1 GENOME RESEQUENCING/ DE NOVO GENOME ASSEMBLY/ GENOME SURVEY SEQUENCES ................................... 6

1.1.2 META RDNA AMPLICON SEQUENCING.................................................................................................................. 6

1.1.3 EXON SEQUENCING/ TARGET REGION SEQUENCING/ WHOLE GENOME SEQUENCING ........................................... 6

1.1.4 WHOLE GENOME BISULFITE SEQUENCING/ GENOTYPING BY SEQUENCING .......................................................... 7

1.1.5 IMMUNE REPERTOIRE SEQUENCING ...................................................................................................................... 7

1.1.6 GENOTYPING ........................................................................................................................................................ 7

1.2 REQUIREMENTS FOR RNA EXTRACTION ............................................................................................................... 8

1.2.1 TRANSCRIPTOME RESEQUENCING/ DE NOVO TRANSCRIPTOME ASSEMBLY/ STRAND SPECIFIC TRANSCRIPTOME . 8

1.2.2 LONG NON-CODING RNA SEQUENCING ................................................................................................................ 8

1.2.3 RNA-SEQ (QUANTIFICATION) ............................................................................................................................... 9

1.2.4 SMALL RNA SEQUENCING .................................................................................................................................... 9

1.2.5 IMMUNE REPERTOIRE SEQUENCING ...................................................................................................................... 9

2 SAMPLE PREPARATION GUIDELINE ..............................................................................................................11

2.1 CELL ................................................................................................................................................................... 11

2.1.1 CELL CULTURE HARVESTING ............................................................................................................................... 11

2.1.2 PRESERVATION METHODS AND TRANSPORT REQUIREMENTS FOR DNA EXTRACTION ........................................... 12

2.1.3 PRESERVATION METHODS AND TRANSPORT REQUIREMENTS FOR RNA EXTRACTION ........................................... 12

2.2 BLOOD ................................................................................................................................................................ 12

2.3 PLASMA/ SERUM ................................................................................................................................................. 13

2.3.1 PREPARATION OF SERUM SAMPLE (WITHOUT ANTICOAGULANT) .......................................................................... 13

2.3.2 PREPARATION OF PLASMA SAMPLE (HEPARIN MUST NOT BE USED) ...................................................................... 14

2.4 ANIMAL TISSUE (EXCEPT ARTHROPOD) .............................................................................................................. 14

2.5 ARTHROPOD ........................................................................................................................................................ 15

2.5.1 LIQUID NITROGEN COLLECTION METHOD ............................................................................................................ 15

2.5.2 COMMERCIAL STABILIZING KIT OR PRESERVING REAGENT .................................................................................. 16

2.6 PLANT ................................................................................................................................................................. 16

2.7 MICROBIOLOGICAL CULTURE ............................................................................................................................. 17

2.8 SALIVA ................................................................................................................................................................ 18

2.9 META SAMPLE ..................................................................................................................................................... 18

2.10 FFPE .................................................................................................................................................................. 20

2.10.1 PRESERVATION METHODS AND TRANSPORT REQUIREMENTS FOR DNA EXTRACTION ....................................... 20

2.10.2 PRESERVATION METHODS AND TRANSPORT REQUIREMENTS FOR RNA EXTRACTION ....................................... 20

2.11 CELL FOR CHIP-SEQ ........................................................................................................................................... 20

2.11.1 CROSS-LINKING CHROMATIN IMMUNOPRECIPITATION .................................................................................... 20

2.11.2 NATIVE CHROMATIN IMMUNOPRECIPITATION ................................................................................................. 21

About This Guideline

This guideline details the requirements and instructions for sending tissue samples for BGI

Scientific Research Services. Please read carefully before collecting tissue samples and sending them

to BGI.

Declaration

For biological agents that require handling in biosafety level 3 or level 4 facilities, purified

nucleic acid samples from those biological agents are acceptable. However, any type of BSL3 or

BSL4 tissue samples will not be accepted. For biological agents that require handling in biosafety

level 1 or level 2 facilities, clients may send tissue samples to BGI only after discussing with our

representatives about pathogenicity and infectiousness of the samples. For any pathogenic or

infectious samples, or potential pathogenic or infectious samples (e.g. tissue, blood, bacterium etc.),

they must be shipped in packaging and clearly labeled on the sample tubes or bags following

instructions below:

Primary receptacles must be leak-proof.

Secondary packaging must be leak-proof and capable of protecting the primary receptacles

during transport conditions.

Enclose a detailed itemized list of contents along with a note stating “Pathogenic substance,

please handle with caution” between the secondary packaging and the outer packaging.

Outer packaging must be rigid and properly labeled.

BGI representatives must notify the sample management center with detailed information

(package tracking number, number of sample, sample type, handling precaution, contact of client,

and estimated arrival time) once the samples are sent.

If you have any questions, please contact our customer service at info@genomics.cn or

telephone 400-706-6615.

Notes and precautions:

The successful rate for DNA/RNA extraction depends on various factors including conditions

during handling and the samples’ characteristics. Therefore, customers must be aware that the quality

of nucleic acid extracted from tissue sample cannot be guaranteed. Please save a backup in case for

re-submission. For samples that are precious or scarce, we do not accept those samples for nucleic

acid extraction considering they are hard to be re-sent. It is suggested to send the extracted nucleic

acid samples instead of tissue samples for precious samples.

a) Timing for freezing tissue sample: once tissue sample is removed from living organism, nucleic

acid, especially RNA, begins to degrade. It is suggested to freeze the tissue sample within 5

minutes after it is removed from living organism. The longer it takes to freeze the sample, the

more likely its nucleic acid degrades.

b) Sample amount: please follow this guideline strictly regarding the amount of sample needed to be

sent. It should be no less than half of the suggested amount in order to extract enough

DNA/RNA. It should also be no more than three times the suggested amount stated in this

guideline since the long time needed to thaw large sample for extraction procedures may lead to

degradation of nucleic acid.

c) Tube for preserving sample: all samples must be sent in centrifuge tubes or screw-capped tubes

following the figure and table below. Do not preserve sample in any kind of sealed bag directly.

Sealed bag will become brittle when froze, which may lead to sample leakage and cross

contamination. Wrapping sample with aluminum foil or other paper is also unacceptable. Packing

sample improperly will lead to failure in extraction experiments.

Tissue Type Tube to Use

Plant and fungi

Animal

Cell and microorganism

Sample for DNA library construction >2K

5 mL screw-capped tube

2 mL screw-capped tube

1.5mL EP tube

2.0mL EP tube

50mL screw-capped tube

d) Sample preservation: freezing sample with liquid nitrogen is always preferred. If liquid nitrogen

is not available, freeze the sample in -80℃ freezer. Commercial nucleic acid preservation

solution can be used following its official handbook. However, preservation solution is not

recommended for plant, fungi, cell, or precious sample.

e) Thawing tissue sample repeatedly: once tissue sample is frozen, the probability of RNA

degradation will be over 80% if sample is thawed repeatedly. The quality of RNA extracted from

repeated thawed sample is unlikely to be qualified for library construction. Please make sure

tissue sample will not be thawed once frozen before it arrives BGI.

f) The quality of RNA extracted from tissue collected over one year ago will be poor. Therefore this

kind of sample is not suggested to be sent for RNA extraction.

g) Other requirements: parts for extraction must be isolated and sent alone (e.g. if extraction is

needed for leaf tissue, stem or root cannot be sent along). Precise selection and accurate weighing

will not be performed during extraction procedures. Extraction using commercial kit is only

suitable for small amount of sample, so it cannot be used for large amount of sample. Appropriate

amount of random part will be taken for extracting nucleic acid qualified for library construction.

If special requirement is needed for extraction, customers should extract and send nucleic acid

sample instead of tissue sample.

1 Sample Requirements

1.1 Requirements for DNA Extraction

1.1.1 Genome Resequencing/ De novo Genome Assembly/ Genome Survey Sequences

1.1.2 Meta rDNA Amplicon Sequencing

1.1.3 Exon Sequencing/ Target Region Sequencing/ Whole Genome Sequencing

Library Type

Tissue Type

DNA Library

(≤800 bp)

DNA Library

(2~10 kb)

DNA Library

(≥20 kb)

PCR Free

(≤800 bp)

Fresh Cell Culture (Number of Cells or

Net Weight) ≥5×106 cells ≥1×107 cells ≥5×107 cells ≥1×107 cells

Fresh Animal Tissue (Net Weight) ≥50mg ≥300mg ≥800mg ≥200mg

Fresh Plant Tissue (Net Weight) ≥200mg ≥800mg ≥1g ≥600mg

Whole Blood (Mammals) ≥1 mL ≥2 mL ≥3 mL ≥2 mL

Whole Blood (Non- Mammals) ≥0.5mL ≥0.5mL ≥1 mL ≥1mL

Thallus (Number of Cell or Net Weight) ≥5×106 cells or

≥200mg

≥2×107 cells or

≥800mg

≥5×107 cells or

≥1×107 cells or

≥800mg

Meta (Stool) ≥600mg — — —

Meta (Soil) ≥600mg — — —

Library Type

Tissue Type Meta rDNA Amplicon Sequencing

Meta (Stool) ≥100mg

Meta (Soil) ≥200mg

Library Type

Tissue Type Exon Target Region WGS

Fresh Cell Culture (Number of Cell or Net Weight) ≥5×106 cells

Fresh Animal Tissue (Net Weight) ≥50mg

1.1.4 Whole Genome Bisulfite Sequencing/ Genotyping by Sequencing

1.1.5 Immune Repertoire Sequencing

1.1.6 Genotyping

Fresh Plant Tissue (Net Weight) ≥200mg

Whole Blood (Mammals) ≥1 mL

Whole Blood (Non- Mammals) ≥0.5mL

Thallus (Number of Cell or Net Weight) ≥5×106 cells or ≥200mg

FFPE ≥ 10 slides, unstained, 100 mm2 area, 5 ~ 10μm

thickness, tumor content≥70%

Library Type

Tissue Type

Whole Genome Bisulfite

Sequencing Genotyping by Sequencing

Fresh Cell Culture (Number of Cell or Net Weight) ≥1×106 cells ≥1×106 cells

Fresh Animal Tissue (Net Weight) ≥50mg ≥50mg

Fresh Plant Tissue (Net Weight) ≥200mg ≥200mg

Whole Blood (Mammals) ≥1 mL ≥1 mL

Whole Blood (Non- Mammals) ≥0.5mL ≥0.5mL

Saliva — ≥1 mL (DNA Genotek

collection kit)

Library Type

Tissue Type Immune Repertoire Sequencing

Library Type

Tissue Type Genotyping

1.2 Requirements for RNA extraction

1.2.1 Transcriptome Resequencing/ De novo Transcriptome Assembly/ Strand Specific

Transcriptome

Library Type

Tissue Type Transcriptome

Fresh Cell Culture (Number of Cell or Net

Weight) ≥2×105 cells

Whole Blood

≥ 1 mL collected lymphocytes from whole blood or ≥ 1 mL Whole

Blood Preserve in Paxgene Blood RNA tube / RNAprotect® Animal

Blood Tubes

FFPE ≥ 5 slides, unstained, 100 mm2 area, 5 ~ 10μm thickness, tumor

content≥70%

1.2.2 Long Non-coding RNA Sequencing

Library Type

Tissue Type LncRNA-Seq

Whole Blood

≥ 1 mL collected lymphocytes from whole blood or ≥

1 mL Whole Blood Preserve in Paxgene Blood RNA

tube / RNAprotect® Animal Blood Tubes

Whole Blood (Non- Mammals) ≥0.5mL

Saliva ≥1 mL (DNA Genotek collection kit)

Thallus (Number of Cell or Net Weight) ≥2×105cell or ≥30mg

1.2.3 RNA-Seq (Quantification)

Library Type

Tissue Type RNA-Seq (Quantification)

Whole Blood

≥ 1 mL collected lymphocytes from whole blood or ≥ 1

mL Whole Blood Preserve in Paxgene Blood RNA

1.2.4 Small RNA Sequencing

Library Type

Tissue Type Small RNA

Whole Blood

mL Whole Blood Preserve in Paxgene Blood RNA

Plasma/ Serum ≥ 1 mL

1.2.5 Immune Repertoire Sequencing

Library Type

Tissue Type Immune Repertoire Sequencing

Whole Blood

mL Whole Blood Preserve in Paxgene Blood RNA tube

/ RNAprotect® Animal Blood Tubes

2 Sample Preparation Guideline

2.1 Cell

2.1.1 Cell culture harvesting

2.1.1.1 Collecting adherent cell by scraping:

a) Discard growth medium;

b) Detach cells by pipetting an appropriate volume of 1X PBS over the cell layer surface several

times. For cells that do not detach easily, scrap the plate with a rubber policeman;

c) Collect the PBS resuspended cells into a screw-capped microcentrifuge tube;

d) Centrifuge the tube at 300-500g (1,100-1,500rpm) for 5 minutes, then discard the supernatant

and collect the cell pellet.

2.1.1.2 Collecting adherent cells by trypsin treatment:

a) Discard growth medium;

b) Gently rinse cells with 1X PBS, then discard the supernatant;

c) Add the pre-warmed dissociation reagent such as trypsin to the side of the flask, use enough

reagent to cover the cell layer (approximately 0.5 mL per 10cm2). Gently rock the container to get

complete coverage of the cell layer;

d) Incubate the culture vessel at room temperature for approximately 2 minutes or until most of the

cells start to detach;

e) Add 1X PBS to disperse the cells by pipetting over the cell layer surface several times;

f) Transfer the PBS resuspended cells into a 15 mL or 50 mL conical tube, then centrifuge at 300-

500g (1,100-1,500rpm) for 5 minutes;

g) Discard the supernatant and collect the cell pellet.

2.1.1.3 Collecting suspension cells:

a) Transfer the cells with growth medium into a 15 mL or 50 mL conical tube and centrifuge at

300-500g (1,100-1,500rpm) for 5 minutes;

b) Discard the supernatant;

c) Rinse cells with PBS, then centrifuge at 300-500g (1,100-1,500rpm) for 5 minutes;

d) Discard the supernatant and collect the cell pellet.

2.1.2 Preservation methods and transport requirements for DNA extraction

2.1.2.1 Liquid nitrogen collection method

a) After collecting cells into a screw-capped centrifuge tube, immediately freeze the sample with

liquid nitrogen and transfer to -80℃ cryopreservation;

b) Ship the sample to BGI along with adequate dry ice.

2.1.3 Preservation methods and transport requirements for RNA extraction

2.1.3.1 Liquid nitrogen collection method

a) After collecting cells into a screw-capped centrifuge tube, immediately freeze the sample with

liquid nitrogen and transfer to -80℃ cryopreservation;

b) Ship the sample to BGI along with adequate dry ice.

2.1.3.2 TRIzol method

a) Alternatively, after collecting cells into screw-capped centrifuge tube, dissolve them in TRIzol

reagent immediately (about 1mL TRIzol lysate for every 1×106 cells);

b) Pipette up and down or vortex intensely to fully resuspend the cell pellet and transfer to -80℃

cryopreservation;

c) Ship the sample to BGI along with adequate dry ice.

Note: It is recommended to freeze freshly harvested tissue with liquid nitrogen within 3 minutes

after the tissue leaves the living body. Long exposure time before freezing will increase the risk of

nucleic acid degradation.

2.2 Blood

2.2.1.1 EDTA tube

It is recommended to use EDTA tubes to collect blood samples (Heparin must not be used as it

will affect the downstream experiment). Ship freshly drawn blood samples to BGI with ice packs,

and frozen blood samples with dry ice (Frozen whole blood samples (EDTA anticoagulant blood

vessels are mostly glass fragile material) are recommended to transfer to 15 / 50ml centrifuge tube

dry ice to prevent EDTA tube in transit to break).

Note: only samples preserved with the three methods below will be accepted by BGI.

2.2.2.1 Separating leukocytes from Whole blood：

a) Add an equal volume of 1X PBS in fresh whole blood sample with anticoagulant (EDTA

recommended, heparin must not be used as it will affect the downstream experiment) and mix it

thoroughly;

b) Slowly transfer it into another centrifuge tube with lymphocyte separating solution, meanwhile,

keep it above the lymphocyte separating solution (do not mix these two types of liquid and keep clear

interface), then centrifuge with 3,000g for 30 minutes;

c) Separate the leucocytes layer with pipette carefully, rinse with 1X PBS, centrifuge to retain the

leukocyte and remove the supernatant;

d) Add Trizol reagent at 20 times volume of leukocyte. Resuspend the cell pellet with a disposable

syringe until there is no visible conglobate cell cluster. The solution should be clear;

e) Transfer to -80℃ cryopreservation, and ship the sample to BGI along with adequate dry ice.

2.2.2.2 Collecting blood samples using PAXgene™ Blood RNA Tubes (16x100 mm / 2.5mL)

a) Product Cat No.: 762165 PAXgene Blood RNA Tube (16x100 mm / 2.5mL);

b) Follow the product manual for collection and shipment instruction.

2.2.2.3 Collecting blood samples using RNAprotect® Animal Blood Tubes (500μL)

a) Product Cat No.: Cat No./ID: 76554 RNAprotect Animal Blood Tubes (50 x 500 µL);

Note: It is recommended to use PAXgene™ Blood RNA Tube for human blood sample.

2.3 Plasma/ Serum

2.3.1 Preparation of serum sample (without anticoagulant)

a) After blood is freshly drawn, let it set at 15-25℃ for 20-60 minutes. When the blood sample has

coagulated completely, centrifuge the sample at 1,600g at 4℃ for 10 minutes;

b) Carefully transfer the supernatant to new tubes, then centrifuge it at 16,000g at ℃ for 10

minutes;

c) Carefully transfer the clear supernatant to nuclease-free screw-capped tubes;

d) Preserve the sample at -80℃ until it is shipped to BGI with dry ice.

2.3.2 Preparation of plasma sample (Heparin must not be used)

a) Use EDTA tube to collect blood sample, then invert it several times to mix the sample with

anticoagulant thoroughly;

b) Centrifuge at 1,600g at 4℃ for 10 minutes. The sample should be separated into three layers:

plasma at the top, leucocytes at the middle, and red blood cells at the bottom;

c) Transfer the top layer plasma to a new tube. Then centrifuge at 16,000g at 4℃ for 10 minutes;

d) Carefully transfer the clear supernatant to nuclease-free screw-capped tubes;

e) Preserve the sample at -80℃ until it is shipped to BGI with dry ice.

Note: The amount of plasma/serum sample should be at least 3mL.

Note: Hemolyzed blood samples should not be sent.

2.4 Animal Tissue (Except Arthropod)

Freshly prepared samples are highly preferred for extraction experiment, while frozen or

dehydrated samples are also acceptable in case of fresh samples are not available. Organs rich in

nucleic acid shall be selected for extraction, for example liver, lung and spleen in animal.

2.4.1.1. Liquid nitrogen collection method

a) Target tissues taken from living bodies should be rinsed with 0.9% DNase-free saline

immediately to remove blood, contaminants and any unneeded tissue;

b) Dry the surface and cut the sample into small pieces around 50 mg (about the size of a soy bean)

on ice. Freeze the tissue sample with liquid nitrogen, then put the specimen into screw-capped

DNase-free tubes;

c) Preserve the sample at -80°until it is shipped to BGI with dry ice.

2.4.1.2. Commercial stabilizing kit or preserving reagent

If commercial stabilizing kit or preserving reagent is used to preserve the samples, please follow

the product manual strictly. Tissues should be kept at size of 5mm to ensure the full penetration of

reagents into samples.

Note: Samples should be frozen with liquid nitrogen within 3 minutes after taken from living

bodies. Long exposure at room temperature will lead to degradation of nucleic acid.

2.4.2.1. Liquid nitrogen collection method

a) Target tissues taken from living bodies should be rinsed with RNase-free water immediately to

remove blood, contaminants and any unneeded tissue;

b) Dry the surface and cut the sample into small pieces around 20-30 mg on ice. Freeze the tissue

sample with liquid nitrogen, then put the specimen into screw-capped RNase-free tubes;

c) Preserve the sample at -80℃ until it is shipped to BGI with dry ice.

2.4.2.2. TRIzol

If TRIzol is used to preserve the sample, please grind the tissue with liquid nitrogen before

transferring it into TRIzol.

a) Grind the tissue with liquid nitrogen before transferring it into TRIzol, Avoid adding excessive

tissue samples, as 10-15mg/mL TRIzol is recommended;

b) Vortex intensely and let it sit at room temperature for 5 minutes for lysis to take place.

c) Preserve the sample at -80° until it is shipped to BGI with dry ice.

2.4.2.3. Commercial stabilizing kit or preserving reagent

If commercial stabilizing kit or preserving reagent such as RNAlater is used to preserve the

samples, please follow the product manual strictly. Tissues should be kept at size of 5mm to ensure

the full penetration of reagents into samples.

Note: Samples should be frozen with liquid nitrogen within 3 minutes after taken from living

bodies. Long exposure at room temperature will lead to degradation of nucleic acid.

2.5 Arthropod

2.5.1 Liquid nitrogen collection method

2.5.1.1. Insect

a) For small insect, such as mosquitoes and ants, freeze the individual with liquid nitrogen and

preserved in screw-capped tubes;

b) For relatively large insect such as caterpillar, rinse the individual with clean water to remove dust.

Then dry the surface and cut the sample into small pieces of around 50 mg (about the size of a soy

bean) on ice. Freeze the pieces with liquid nitrogen and put in screw-capped tubes;

2.5.1.2. Crustacean (crabs, shrimps, etc.)

a) Rinse the living crustacean organism with clean water, and dry the surface;

b) Dissect the individual to separate the meat from the shell. Cut the meat tissue into small pieces of

around 50 mg (about the size of a soy bean) on ice. Freeze the pieces with liquid nitrogen and put in

screw-capped tubes;

2.5.2 Commercial stabilizing kit or preserving reagent

2.5.2.1. Insect

a) Rinse the living crustacean organism with clean water, and dry the surface. Follow the product

manual strictly to preserve the sample. Tissues should be kept at size of 5mm to ensure the full

penetration of reagents into samples;

b) Preserve the sample at -80℃ until it is shipped to BGI with dry ice.

2.5.2.2. Crustacean (crabs, shrimps, etc.)

a) Rinse the living crustacean organism with clean water, and dry the surface;

b) Dissect the individual to separate the meat from the shell. Cut the meat tissue into small pieces on

ice. Then follow the product manual strictly to preserve the sample. Tissues should be kept at size of

5mm to ensure the full penetration of reagents into samples;

Liquid nitrogen collection methods is preferred if it is available.

2.6 Plant

Freshly prepared samples are highly preferred for extraction experiment, while frozen

ordehydrated samples are also acceptable in case fresh samples are not available. Organs rich in

nucleic acid shall be selected for extraction, such as young organs in plants.

a) Samples collected from the field and countryside need to be rinsed by 70% ethanol for DNA

extraction and RNase-free water for RNA extraction to remove dust and mud;

b) Dry the surface and cut the sample into small pieces around 50-100 mg on ice. Freeze the tissue

sample with liquid nitrogen, then put the specimen into screw-capped nuclease-free tubes;

If commercial stabilizing kit or preserving reagent is used to preserve the samples, please follow

the product manual strictly. Tissues should be kept at size of 5mm to ensure the full penetration of

reagents into samples.

Note: In order to reduce the oxidation of secondary metabolites, samples rich in polysaccharide/

polyphenol should be kept at -80°, or frozen in liquid nitrogen then stored in -80℃ once collected. A

trace amount of deoxidizer (e.g. PVP) can be added to the samples to prevent oxidation of

polyphenol. It is also recommended to place the plant in dark for 24-48 hours before collecting

samples, in order to reduce the amount of secondary metabolite in samples. However, the treatment

may not be effective for all kind of plants.

Samples should be frozen with liquid nitrogen within 3 minutes after taken from living bodies.

Long exposure at room temperature will lead to degradation of nucleic acid.

Liquid nitrogen collection methods is preferred if it is available.

2.7 Microbiological Culture

All microorganism sample must be isolated from culture medium. Do not send culture medium

along with the sample, which will not be accepted for extraction experiments.

a) Collect the microorganism sample culture in a 15/50mL tube. Centrifuge and discard the culture

medium;

b) Rinse the samples by bacteria-free water or PBS for 1-3 times. Then resuspend the sample pellet

and transfer it to a nuclease-free screw-capped tube. Centrifuge and discard the supernatant;

If commercial stabilizing kit or preserving reagent is used to preserve the samples for

DNA&RNA extraction, please follow the product manual strictly.

Note: RNAlater is not recommended for preserving microorganism sample, because RNAlater is

so dense that it will make it difficult to collect the sample through centrifugation.

2.8 Saliva

Commercial stabilizing kit or preserving reagent

a) Use Oragene•DISCOVER (OGR-500) (For Research) or Oragene•Dx (OGD-500) (For

Diagnostics) collection kit from DNA Genotek Inc.;

2.9 Meta sample

For the meta sample extraction effect, depending on the microbial abundance of the sample, the

low abundance of the sample may not be able to extract nucleic acids.

2.9.1.1. Stool sample from human and large animal

a) Prepare a clean container for collecting the sample;

b) Use a clean toothpick or any commercial collection device to take a small piece (about the size

of a peanut, no larger than 1/3 volume of a 2mL centrifuge tube) of sample from the middle part of

the specimen. The outer part of the specimen is easily contaminated, and the nucleic acid also

degrades quickly once the sample comes into contact with air. Put the sample in a clean screw-

capped tube. It is recommended to prepare 3-5 copies for each sample;

c) Freeze the sample immediately. Preserve the sample at -80℃ until it is shipped to BGI with dry

2.9.1.2. Stool sample from small animal (e.g. mouse)

a) Collect the granular specimen immediately after defecation. Put the sample in a clean screw-

capped tube (about three pieces per tube for mice);

b) Freeze the sample immediately. Preserve the sample at -80℃ until it is shipped to BGI with dry

2.9.1.3. Intestinal content

a) Dissect the target intestine part in a sterile operation condition;

b) Collect the intestinal content (about the size of a peanut, no larger than 1/3 volume of a 2mL

centrifuge tube) using a clean scalpel. Put the sample in a clean screw-capped tube. It is

recommended to prepare 3-5 copies for each sample;

2.9.1.4. Microorganism from intestinal tissue

a) Rinse the sample with 1xPBS to remove the intestinal content;

b) Scrap the inner surface with sterile glass slide, then transfer the sample to a clean 2mL centrifuge

2.9.1.5. Soil

a) Determine the sampling location according to the research purpose. Sterilize all the collection

instruments and consumables;

b) Use appropriate tools to collect sample from the surface down to about 5-20cm.

c) Remove most of the visible impurities. Filter the sample with 2mm-pored filter. It is

recommended to collect and combine soil from three spots for each sample. Collect the soil sample

(about the size of a peanut, no larger than 1/3 volume of a 2mL centrifuge tube) in a clean 2mL

screw-capped tube. It is recommended to prepare 3-5 copies for each sample;

d) Freeze the sample immediately. Preserve the sample at -80℃ until it is shipped to BGI with dry

2.9.1.6. Meta water samples

a) Determine the sampling location and depth according to the research purpose;

b) Filter the water ample using filter paper with appropriate pore size;

For human stool sample, use OMNIgene•GUT (OMR-200) collection kit from DNA Genotek

Inc. Follow the product manual for collection and shipment instruction.

2.10 FFPE

FFPE slides should be at least 10 unstained slides of 5-10μm. The slides should contain tissue

area of at least 100 mm2 in which more than 80% are nucleated cells and more than 70% are cancer

cells. FFPE sample can be shipped at room temperature.

FFPE slides should be at least 5 unstained slides of 5-10μm. The slides should contain tissue

area of at least 100 mm2 in which more than 80% are nucleated cells and more than 70% are cancer

cells. FFPE sample can be shipped at room temperature.

2.11 Cell for ChIP-Seq

For ChIP-Seq cell samples: At least 5×107 cells are required for one sample preparation (for

human or mouse cell line, library construction can be tried with 1×107 cells, however, more cells are

preferred). Total cell number for multiple preparations equals to number of sample preparation×5

×107. Customers should also offer a small amount of sample (1/10 of total samples) in a separate

tube for early detection. Special category tissues are required to submit an extraction consultation in

advance.

2.11.1 Cross-linking Chromatin Immunoprecipitation

a) Prepare the 1% formaldehyde with PBS (pH7.4). Pre-warm in a water bath to 37℃;

b) Collect the cells in 15mL centrifuge tubes. Remove culture medium without disturbing the cell

pellet by centrifugation at 850g for 5min. Crosslink by adding 1% formaldehyde and incubate at 37

℃ for 10min, mix for every 3-5min;

c) Stop the cross-linking by adding glycine to a final concentration of 125mM. Continue to

incubate at room temperature for 5 min;

d) Wash the cell with ice-cold 1×PBS twice. Then add 1mL [1×PBS plus 1xProtein Inhibitor

Cocktail] to the sample. Transfer the sample to 1.5 mL EP tube, centrifuge at 850g for 5min to

remove the PBS cocktail;

e) Freeze the samples in liquid nitrogen. Preserve the sample at -80℃ until it is shipped to BGI

with dry ice.

2.11.2 Native Chromatin Immunoprecipitation

a) Remove culture medium without disturbing the cell pellet by centrifugation at 850g for 5min;

b) Wash the cell with ice-cold 1×PBS twice. Then add 1mL [1×PBS plus 1xProtein Inhibitor

Cocktail] to the sample. Transfer the sample to 1.5 mL EP tube, centrifuge at 850g for 5min to

remove the PBS cocktail;

c) Freeze the samples in liquid nitrogen. Preserve the sample at -80℃ until it is shipped to BGI

with dry ice.

Tissue Sample Submission Guideline - BGI A0 Tissue Sample... · Blood Preserve in Paxgene Blood RNA...

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Transcript of Tissue Sample Submission Guideline - BGI A0 Tissue Sample... · Blood Preserve in Paxgene Blood RNA...

Blood…why? Connective Tissue Plasma White blood cells 55 m Red blood cells Cartilage Chondrocytes Chondroitin sulfate 100 m Adipose tissue Fat droplets.

DNeasy Blood & Tissue Handbook - Qiagen

Blood. Blood - Introduction Connective Tissue inside blood vessels Connective Tissue inside blood vessels.

DNeasy Blood & Tissue Handbook - Home ExD · PDF file6 DNeasy Blood & Tissue Handbook 07/2006. ... The following risk and safety phrases apply to components of DNeasy Blood & Tissue

Ordering Information — QIAGEN Promotion March 2017 · Ordering Information — QIAGEN Promotion March 2017 Until March 24, ... 76526 RNAprotect Cell Reagent (250 ml) 76544 RNAprotect

€¦ · Web viewConnective Tissue Epithelial and Glandular Tissue Nervous Tissue Muscle Tissue Connective Tissue Healing Some connective tissues have a limited blood supply Prolonged

Control of Tissue Blood Flow 19 Med

Supporting Text Blood and Tissue Processing. Blood and ...€¦ · Supporting Text Blood and Tissue Processing. Blood and tissue samples were processed immediately after collection.

1 Control of blood tissue blood flow Faisal I. Mohammed, MD,PhD.

Blood, Tissue Fluid and Lymph

05 blood liquid connective tissue

Blood and Haemopoiesis. Overview of Blood Blood is a fluid connective tissue. Blood is a fluid connective tissue. Its total volume is about 6 liters.

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IB Blood. Blood Blood is a connective tissue that contains both dissolved substances and specialized cells.

Body Tissue Epithelium and Blood

DNeasy Blood & Tissue Handbook

Pa Blood Tissue Protozoa Dogs Cats

Chapter 10: Blood. Blood The only fluid tissue in the body Classified as a connective tissue Notable: collagen & elastin absent from blood Dissolved.

Blood Tissue

Recombinant Tissue Plasminogen Activator Increases Blood ...