TITLE: Collecting needle and branch samples for terpenoid and expression analysis
AUTHORS: Vidya S Vuruputoor, karlfetter, victoriaburton, Jill Wegrzyn

[DESCRIPTION]
This protocol is used by the PCG group for sampling needle and branch tissue from hemlock conifers for terpenoid analysis, as well as RNA expression analysis.

[STEPS]
SECTION: TreeSnap Observation
1. Open Treesnap (https://treesnap.org/), record the accession number on the tree and fill in notes for hemlock woolly adelgid infestation (HWA) levels (scale provided by the app).

  

SECTION: TreeSnap Observation
1.1. Use the comment field to enter: EHS present OR EHS absent to record the hemlock scale.

  

SECTION: TreeSnap Observation
1.2. Take at least 3 photos with TreeSnap. This includes at least one of the trees itself, one photo of the tree tag if that is present, and one photo of the collection vial with the label as well.
SECTION: TreeSnap Observation
1.3. Geolocation will be recorded automatically but TreeSnap can also connect with a more precise external GPS device.
SECTION: TreeSnap Observation
1.4. Note DBH in appropriate column
SECTION: TreeSnap Observation
1.5. Note any other observation you may have- including plant condition (Excellent/Good/Poor), and placement of individual (lower canopy, full sun, etc.)
5. RNA- You would use one 5ml vial. For this, 2 branches would be more than sufficient to fit into the vial. Ideally, you would want to sample from 2 different locations of the individual. 

 

4. Metabolomics- You would use 2 10ml vials. You will be able to fit 4 branches (5cm) in each tube- so sample from one direction of the tree, then collect the three other branches from the other sides of the tree (you would ideally try to sample around the individual)
SECTION: Placing in Liquid Nitrogen
6. Close cryovials, and place the vial slowly into the LN dewar. Be wary of LN splashing out, and wear appropriate gear, and stay at a distance.

 

SECTION: Cleaning before sampling from new individual
7. Clean trimmers between trees with ethanol (spray bottle).

 

SECTION: Cleaning before sampling from new individual
8. Use a clipboard to manually record notes on a pre-printed spreadsheet with sample names.
SECTION: Cleaning before sampling from new individual
9. After returning from the field, ensure that notes are consistent with TreeSnap entries.  These entries can be amended after collection as needed.
SECTION: Collection
3. You will collect two sets of samples from each tree. One for RNA extraction for transcriptomics and one for metabolomics.  The first needs less material so we will use the 5ml tubes.  The latter needs more material and will use the 10ml tubes.  The first should include two branches that are approximately 5 cm in length.  The second should include four branches, ideally taken from different locations around the tree

 

SECTION: Prepare sampling sheet
2. The sample sheet would look like this:- Link to spreadsheet
SECTION: Post-sampling and Pre-shipping
10. Remove all vials from the dewar (cross-check with the sample sheet). Samples must be stored at -80C or should be on dry ice during transport.

Cross-check all samples at this stage with your sample sheet.
SECTION: Shipping
12. Log all data and samples in a Google sheet, or send over sample sheet so that we log your data
SECTION: Shipping
11. Shipping instructions will be provided via email. Shipments must be sent on dry ice. You will be shipping separate parcels for the Metabolomics and RNA samples.