TITLE: Biospecimen Aliquotting
AUTHORS: Clemens Scherzer, Bradley Hyman, Charles Jennings

[DESCRIPTION]
This protocol explains the Standard Operating Protocol for aliquotting Bio-Specimens.

[GUIDELINES]
FREEZER STORAGE
  
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.

[STEPS]
SECTION: 1-2 days before aliquotting:
1. Order dry ice the night before, at the latest, for delivery at 9 am.
SECTION: 1-2 days before aliquotting:
2. Compile list of parent aliquots according to freezer position. Group aliquots from the same rack, column and box in the freezer.
SECTION: 1-2 days before aliquotting:
3. Pull parent aliquots from separate locations and pool into one or more boxes. Place in one location in the freezer.
SECTION: 1-2 days before aliquotting:
4. Print labels and label tubes for sub-aliquots.
SECTION: 1-2 days before aliquotting:
5. In order to save time, scan barcodes of sub-aliquots into freezerworks.
SECTION: Day of aliquotting:
6. Place parent aliquots on ice to thaw for about 120 min.

SECTION: Day of aliquotting:
7. Sub-aliquotting parent aliquots:
SECTION: Day of aliquotting:
7.1. Pipette samples in numerical order by sub-aliquot barcode label.
SECTION: Day of aliquotting:
7.2. Any sample left over in the parent tube should be distributed evenly amongst the subaliquots.
SECTION: Day of aliquotting:
7.3. Extra sub-aliquots can be created if remaining amount of sample in parent tube equals or exceeds desired volume.
SECTION: Day of aliquotting:
7.4. Do not discard parent tube. Place in tube rack and set aside for scanning.
SECTION: Day of aliquotting:
7.5. Do not discard extra, empty sub-aliquot tubes. Place in tube rack and set aside for scanning.
SECTION: 1-2 days before aliquotting:
5.1. Highlight parent aliquot to be sub-alquotted. 
SECTION: 1-2 days before aliquotting:
5.2. Click sub-aliquot. 
SECTION: 1-2 days before aliquotting:
5.3. Type in number of sub-aliquots, volumes (current and initial), freeze thaws, and amount to be deducted from parent aliquot. 
SECTION: 1-2 days before aliquotting:
5.4. Scan sub-aliquots into freezerworks. 
SECTION: 1-2 days before aliquotting:
5.5. Position sub-aliquots (position one sub-aliquot in parent aliquot’s original position).
SECTION: Day of aliquotting:
8. Place one new sub-aliquot in the original position previously held by the parent aliquot.
SECTION: Day of aliquotting:
9. Place remaining sub-aliquots immediately into the -80 °C freezer in positions determined in Step 5.5.

SECTION: Day of aliquotting:
10. Scan parent aliquot in “select by scanned field” function. Update parent aliquots by setting “current amount” to 0. 
SECTION: Day of aliquotting:
11. Scan extra, empty sub-aliquots in “select by scanned field” function. Delete aliquots entirely.
SECTION: Day of aliquotting:
12. Add transactions in Freezerworks for each aliquot given to a collaborator outlining:
SECTION: Day of aliquotting:
12.1. Date of aliquotting.
SECTION: Day of aliquotting:
12.2. Type of transaction (i.e. “imparting of specimens”).
SECTION: Day of aliquotting:
12.3. A note that describes to whom the aliquot is for, the volume, and type of aliquot.
SECTION: Day of aliquotting:
12.4. (see “Adding transactions” protocol for additional information.)