TITLE: Sanger Tree of Life Sample Preparation: Triage and Dissection
AUTHORS: Jessie Jay, Halyna Yatsenko, Juan Pablo Narváez-Gómez, Haddijatou Mbye, Maria Morra, Michelle Strickland, Clare Cornwell, Caroline Howard

[DESCRIPTION]
This protocol describes the process of sample preparation for the extraction of DNA and/or RNA from the wide variety of samples processed by the Tree of Life Core Laboratory as part of the Tree of Life Programme. It also describes the triage steps and recommended weight requirements for the different taxonomic groups covered by the Tree of Life programme. The output of this protocol is a sufficient amount of sample that can be directed towards any of the Sanger Tree of Life Sample Homogenisation protocols.

[BEFORE_START]
Prepare at least 2 rubber buckets with dry ice (use more depending on the number of samples and other requirements/ personal preference):
Cover one dry ice bucket with aluminium foil and place a metal dissection plate on top.
Use the second dry ice bucket to store the samples that are going to be prepared. Samples should be kept on dry ice to ensure they remain frozen.
Clean and pre-chill materials needed for dissection (weigh boats, scalpel, tweezers, scissors, etc.) by placing them on dry ice.
Set up analytical balance.
Retrieve frozen samples. 

[GUIDELINES]
Sample triage consists of weighing the samples, dissecting and aliquoting portions of samples as needed. Recommended weights can be found in Table 1, and are determined by sample type, downstream process and weight. More in depth decision trees can be found in the Appendix. 
The majority of samples submitted to the Sanger Tree of Life are those that have been snap-frozen; these are processed as ‘standard samples’. However, samples which have not been snap-frozen are also submitted; these samples include blood samples preserved in ethanol and tissues stored in preservative solutions such as RNAlater, DMSO, DESS & ethanol. These samples are referred to as ‘non-standard samples’ and they have varying degrees of success downstream of sample preparation.
Hi-C processing is completed by the Long Read Sequencing Team within DNA Pipelines Operations at the Wellcome Sanger Institute, therefore within this protocol, the required tissue prep refers to the sample which is sent for further processing outside of the Tree of Life Core Laboratory.
It is essential that samples remain frozen throughout this process for high quality extraction and sequencing results:
A cold rack can be used to keep samples cold and organised.
Tools used for dissection should be pre-chilled and kept in dry ice when not in use (tweezers, scalpel). 
Samples should be dissected on a pre-chilled metal dissection plate. 
Try to minimise the time that the sample is not on dry ice (i.e. when weighing on the balance). 


Table 1. Tissue preps required by downstream process
   Downstream processRequired tissue prepRecommended Tissue weight before CryoprepHiCUp to 20 mg of frozen tissue for Metazoa can be sent as whole frozen tissueOR 50 mg of cryoprepped tissue for all taxonomic groups (excluding protists) If the sample is an arthropod and the head is visible, dissect and take the head onlyMetazoa: 60-70 mgPlants/Algae/Fungi: 60-70 mgProtist: Never CryoprepDNA Extraction - MagAttract/Plant MagAttractUp to 25 mg of frozen tissue for Metazoa can be powermashedORCryoprepped tissue weight depends on taxonomic group requirements:Metazoa: 25 mgPlants/Algae/Fungi/Corals: 50 mgProtist: 50 mgMetazoa: 30-40 mgPlants/Algae/Fungi/Corals: 60-70 mgProtist: 55-100 mgDNA Extraction - Plant Organic Extraction (POE)65 mg of cryoprepped plant/fungi Plants: 80-90 mgIf less than 40 mg do not proceedRNA ExtractionUp to 15 mg of frozen tissue for Metazoa can be powermashedORCryoprepped tissue weight depends on taxonomic group requirements:Metazoa: 15 mgPlants/Algae/Fungi/Corals: 15-20 mgMetazoa: 20-30 mgPlants/Algae/Fungi: 20-30 mg

 Additional Notes
Protist samples can be pooled together if from the same colony in order to reach the recommended weight.
Samples that have shells and which do not weigh enough to be prepared by cryoPREP are not recommended for further processing. The shell makes the sample difficult to powermash, and tissue loss after cryoPREP will result in not not enough remaining tissue for the downstream process.
 

Appendices:
DNA Decision Making Tree:   
RNA Decision Making Tree:   
HiC Decision Making Tree:   

[STEPS]
SECTION: Laboratory procedure
1. Select the first sample from the cold rack.
SECTION: Laboratory procedure
2. Inspect the sample and check the sample preservation method. 
For snap frozen samples (‘standard samples’), proceed to step 3. 
For samples that have not been snap frozen (‘non-standard samples’), please refer to step 7. 
SECTION: Laboratory procedure
3. Decide how much sample is required for the desired downstream process (see Table 1 or the relevant Decision Making Tree in Appendices). 
SECTION: Laboratory procedure
4. Weigh the sample. If taking only part of a sample, dissect the desired amount. 
Use an analytical scale to tare a new clean weighing boat. 
Use clean and pre-chilled dissection tools (scalpel, tweezers, scissors) and metal dissection block to cut the sample until the desired weight has been achieved. 
Place prepared sample into correct destination container and put back any remaining tissue into original container
Clean the tools and dissection block with Azowipe tissue and bury the dissection tools in dry ice so that they are ready for the next sample.
SECTION: Laboratory procedure
5. If the total weight is 25 mg or less and it is from Metazoa (excluding sponges and corals), use the ‘Sanger Tree of Life Sample Homogenisation: PowerMash’ protocol. 
SECTION: Laboratory procedure
6. If the total weight is greater than 25 mg for all taxonomic groups, use ‘Sanger Tree of Life Sample Homogenisation: Covaris cryoPREP® Automated Dry Pulverizer’ protocol. 
SECTION: Laboratory procedure
7. The current method to prepare samples stored in preservative solutions is as follows:
Place the tube containing the sample in wet ice to allow the liquid to defrost. 
Remove the sample from solution and transfer to a new tube. Dispose of the preservative solution in an appropriate waste container.
Then proceed as if the sample was frozen tissue (see step 3). Keeping in mind the recorded weight will not be accurate, as the sample will have absorbed some of the preservative solution.
SECTION: Laboratory procedure
8. If the sample type is blood, defrost on wet ice and aliquot required amount for the desired downstream protocol. If the blood is stored in ethanol, the aliquot should be taken from underneath the layer of ethanol.