PacBio Tissue Sample Submission Guideline

Transcription

1 PacBio Tissue Sample Submission Guideline Document NO.: SOP-SMM-020 Version NO.: A1 Effective Date:

2 Document NO.:SOP-SMM-020 Version NO.:A1 Page 1of 12 Directory 1 OBJECTIVES SCOPES OPERATING PROCEDURE PACKAGING AND DELIVERY RELATED DOCUMENT RELATED RECORDS REFERENCE STANDARDS AND DOCUMENTS... 12

3 Document NO.:SOP-SMM-020 Version NO.:A1 Page 2of 12 1 Objectives This submission guideline is written to provide PacBio tissue sample preparation guidance for relative customers to improve success rates of sample extraction. 2 Scopes Collection and processing of PacBio samples should follow the procedure provided in this guideline before sending to BGI for tissue extraction. 3 Operating procedure 3.1 Amount needed of tissue Table 1:Amount needed of different tissue types Tissue types Dry weight of fresh animal tissue Amount needed for DNA extraction 1.5-5g Amount needed for RNA extraction mg Dry weight of fresh plant tissue 5-10g mg Fresh cultured cells cells cells Whole blood (Mammals) 5-10 ml 2-5mL Whole blood (Non-mammals) 2-5mL 0.5-2mL Bacteria cells cells Algae cells or 5-10g cells or 1-2g P.S. Sufficient amount of tissue is required but sending excessive tissue will hinder tissue extraction and storage. 3.2 Operating procedure of sliced tissue using liquid nitrogen flash-freezing technique

4 Document NO.:SOP-SMM-020 Version NO.:A1 Page 3of Prepare thermal container (referred to Picture 1),germ-free aluminum container or similar plastic box (Picture 2),Ladle,forceps,scissors,scalpel screw cap tubes etc.. Picture 1. Thermal container Picture 2. Aluminum container Transfer appropriate amount of liquid nitrogen to thermal container and prepare for the tissue sample. Add appropriate amount of liquid nitrogen as to pre-cool the aluminum box Cut appropriate amount of tissue sample and wash it quickly with nuclear-free water and dry it with absorbent paper. Then cut it into smaller pieces and put it immediately into the aluminum box with liquid nitrogen. Refill the aluminum box with liquid nitrogen before it evaporates completely and repeat the procedure until the tissue sample is completely frozen Transfer the tissue sample to a screw cap tube by forceps when it is completely frozen. Store it in -80 fridge or thermos bottle with liquid nitrogen immediately to prevent thawing P.S: Large pieces of tissue sample need to be cut smaller and lump-like tissue should be frozen by direct contact with liquid nitrogen.

5 Document NO.:SOP-SMM-020 Version NO.:A1 Page 4of Demonstration of crew cap tube for storing tissue sample Picture 3. Types of screw cap tubes recommended. Picture 4. Types of screw cap tubes unrecommended Picture Collections of different types of tissue and precautions Plant tissue

6 Document NO.:SOP-SMM-020 Version NO.:A1 Page 5of Select healthy part of the tissue sample with a high priority, for example young leaves and young stems Follow procedure 3.2 for operating procedure of sliced tissue using liquid nitrogen flash-freezing technique, store the sample by using screw cap tube. Do not use EP tube instead, as it is easily exploded by the sudden expansion of evaporation of liquid nitrogen. Consequently, sample will be lost. Plant tissue folded with aluminum foil can also be frozen and should be put into a small sealed plastic bag one by one. Do not put more than one sample into a sealed plastic bag to prevent cross contamination. Do not put the tissue sample into the sealed plastic bag without aluminum foil folding as the plastic bag might become fragile after directly contacting with liquid nitrogen(referred to Picture 6). Sample will be lost and cross contamination will be occurred as a result. Picture 6 Note: When liquid nitrogen is used to quickly freeze plant tissue, be sure to wipe the moisture on the surface of the plant tissue (to avoid DNA degradation caused by freeze-thaw during sample transport), and try to send it to a large tube or foil sent Animal tissue Wash the tissue with saline or fresh water to remove blood and impurity after taking out from the original tissue. Dry it with absorbent paper. Then cut the tissue sample into soybean-sized pieces and follow the procedure 3.2 for operating procedure of sliced tissue using liquid nitrogen flash-freezing technique.

7 Document NO.:SOP-SMM-020 Version NO.:A1 Page 6of Collect blood sample for DNA extraction into the EDTA anticoagulation tube. Invert the tube several times. Freeze it by using liquid nitrogen or store it into -80 fridge after anticoagulation. Picture 7 Note: Please send the anticoagulant tube with waste newspaper or paper in the mailing bag when sending, in order to avoid the sample contamination caused by crushing and damage during the sending process, as shown in Picture Collect blood sample for RNA extraction into the EDTA anticoagulation tube. Invert the tube several times and let stand for 5 minutes for anticoagulation purpose. Add red blood cell lysis buffer to remove red blood cell and collect lymphocytes for storage. Freeze it by using liquid nitrogen or store it into -80 fridge after anticoagulation. Or use specific blood RNA protecting tube, BD PAXgene Blood RNA Tube (762165), to collect and store blood. Avoid using EDTA anticoagulation tube to collect blood and freeze directly, as it will cause RNA degradation Algae tissue Use 0.3M EDTA to wash algae tissue after tissue collection. Then flash-freeze it using liquid nitrogen and store it into -80 fridge after the tissue is frozen thoroughly. Note: Be sure to remove as much moisture from the sample as possible Thallus Samples

8 Document NO.:SOP-SMM-020 Version NO.:A1 Page 7of Collect thallus colony in log phase of the bacterial growth cycle Collect pure thallus colony by removing impurities such as the culture media before sending samples Storage of fungi cultivated using liquid culture media. According to the density of fungi, collect appropriate amount of fungi tissue using screw cap tube. Centrifuge in room temperature with 5000 rpm 5 minutes. Remove supernatant and add sterile water or PBS to wash the precipitate. Centrifuge in room temperature with 5000 rpm 5 minutes again. Screw the tube tightly, the bacteria were transferred to a large centrifuge tube and snap frozen in liquid nitrogen, and store it into -80 fridge after the fungi sample is completely frozen. Then sent to BGI, and the same sample was not sent in multiple tubes, as shown in Picture 8. To ensure the pre-experiment and experiment proceed smoothly 2-3 tubes to send; information sheet must fill in the sample information clearly, such as negative bacteria, positive bacteria, fungi, etc Storage of fungi cultivated using solid culture media. Obtain fungi sample by scratching the fungi located on the surface of the solid culture media using spatula or other suitable tools and transfer the sample into a screw tube. Avoid scratching the surface of the media. Screw the tube tightly, put it into a thermal container filled with liquid nitrogen to freeze the sample, and store it into -80 fridge after the fungal sample is completely frozen. Picture 8. Sample which is collected and stored bad (left) and well (right)

9 Document NO.:SOP-SMM-020 Version NO.:A1 Page 8of Avoid sending thallus samples with nutrient agar or broth. Samples without freezing and sending with culture media will be easily contaminated with other thallus species during transport or thallus will enter death phase before extraction, which will cause DNA/RNA degradation, as shown in Picture 9/10. Sending frozen samples with culture media will also cause DNA/RNA degradation during thawing of frozen samples before extraction. Picture 9(left). Contamination of fungi sample sent with culture medium Picture 10(right). Molding and rotting of fungi sample sent in room temperature Storage of large pieces of fungi such as mushroom or ganoderma should follow the procedure described in Special sample delivery diagram and Precautions when sending samples Polysaccharide Polyphenols Samples such as Grape, Azalea, Rhododendron, Artocarpus altilis, Tobacco (partial), Dendrobium nobile Lindl, Tomato, Sesamum indicum, Cherry, Zanthoxylum bungeanum Maxim etc. Samples of high wax, Sorghum bicolor (L.) Moench, Ficus pumila Linn., Castanopsis fargesii Franch., Quercus spinosa, etc. Samples with high starch content such as Cycas panzhihuaensis L. Zhou et S. Y. Yang, etc. Because the extraction yield is lower than the

10 Document NO.:SOP-SMM-020 Version NO.:A1 Page 9of 12 conventional sample, it needs to send 2-3 times the amount of the conventional sample to ensure the success of follow-up experiments and the implementation of optimized solutions. 4 Packaging and Delivery 4.1 Information sheet submission Click in and choose option "tissue sample information sheet" to provide sample's information. Specific manipulation should refer to BGI, Sample Submission Guideline (NGS) chapter 4 "sample packaging and delivery". The information sheet or at least, the batch number, should be printed out after submission of information in order to send together with the sample package. Each sample and information sheet should be packed individually in a package (referred to Picture 11, 12 and 13) to prevent cross-contamination due to sample leakage. Picture 11. Individually packed sample Picture 12. Individually packed information sheet

11 Document NO.:SOP-SMM-020 Version NO.:A1 Page 10of 12 Picture 13. Each individually packed sample and information sheet should be sent in one package Please provide details related to the sample (referred to Picture 14), such as species name (in Latin), methods of processing before obtaining sample etc., so that the sample can be sufficiently understood and increase success rate of extraction. If you have tried to extract by yourself, please describe the extraction procedure and result in details.

12 Document NO.:SOP-SMM-020 Version NO.:A1 Page 11of 12 Picture 14. Information of sample (in red frames) should be provided in details 4.2 Packaging Prepare sample in accordance with 3.2/3.3 and seal the tube with parafilm. It is necessary to wrap the tube with bubble wrap or cotton and separate each tube in order to prevent breaking of tube due to collision during transport (referred to Picture 15.). Instead of glass tubes, plastic tubes are recommended to prevent tubes breakage caused by vigorous vibration during transport or low temperature environment created by dry ice.

13 Document NO.:SOP-SMM-020 Version NO.:A1 Page 12of 12 Picture 15. Wrap the tube with bubble wrap or cotton 4.3 Sample labeling All samples must be labeled clearly and same as the information provided in the information sheet. Labels on cap, tube and package should be the same. Make sure labels written by oil-based paint marker or label stickers are water-proof and can withstand low temperature Samples with the same color with dry ice or relatively small-sized samples should be tagged so that they can be obviously seen to prevent missing samples. 5 Related Document 5.1 SOP-SMM-003 Sample Submission Guideline (NGS) 6 Related Records No 7 Reference Standards and Documents No