TITLE: Automated DNA/RNA Extractions from a stony coral (Acropora palmata) using ZymoBIOMICS DNA/RNA Magbead Kit and the Kingfisher Flex 
AUTHORS: Benjamin   Young

[DESCRIPTION]
This is the protocol used to extract DNA and RNA from the same piece of coral tissue and skeleton using the Zymo DNA/RNA MagBead and ZymoBIOMICS DNA/RNA MagBead kits with automation on the KingFisher Flex. Protocol goes from coral sampling and storage to sample prep for the DNA and RNA protocols on the KINGFISHER Flex. 

[STEPS]
SECTION: Coral Tissue Sampling and Storage
1. For each sample, bonecutters and tweezers were cleaned with 80% bleach and then flame sterilized with 70% ethanol. The bench space was also cleaned between each sample with 80% bleach and 70% ethanol. 
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
3. Thaw stored coral samples at room temperature for 45 minutes. 
During this time prep Zymo bead beating tubes (Zymo Bashingbead Lysis Tube) by filling with 750µl DNA/RNA shield.
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
4. Remove coral skeleton from cryo vial tubes and place in Zymo bead beating tubes filled with 750µl DNA/RNA shield. Bleach and flame with 70% ethanol tools between each coral sample. 
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
5. Using a Genie 2 vortex with 24 tube holder attachment (Vortex 24 Tube Attachment), bead beat 18 tubes MAX (per 24 tube holder attachment) for 30 minutes. 18 samples per attachment is to maintain as high a vortex speed as possible. 
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
6. Centrifuge bead beating tubes for 30 seconds at 10,000g, and transfer 200µl of each sample to a KingFisher deep well plate (LINK). This is the sample plate. 
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
7. To each well with 200µl supernatant, add
- 10µl reconstituted Proteinase K
- 500µl DNA/RNA lysis buffer
- 30µl ZymoBIOMICS MagBinding beads. 
SECTION: Coral Tissue Sampling and Storage
2. All coral samples were stored in 2ml cryo vial tubes (Thermofisher Nalgene 2ml Cryo Vial Tubes) with 1.5ml of RNA later (Thermofisher RNAlater) at -80oC. 
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
6.1. Bead beating tubes with remaining 550µl DNA/RNA shield, beads and coral tissue can be restored at -80oC indefinitely for future use. 
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
5.1. During bead beating step, prep the consumables and reagents needed for the Kingfisher DNA parallel protocol. 
DeepWell plates - 
Tip plate - 
Tip comb - 
  
Make sure all reagents are prepared accordingly following ZymoBIOMICS protocol
- DNA/RNA Wash 1 and 2 are concentrates and require addition of isopropanol. 
SECTION: Tissue Homogenization and Prep for Kingfisher Flex
5.2. Reconstitute lyophilized Proteinase K following ZymoBIOMICS protocol. Store aliquots at -20oC
Reconstitute DNASE following ZymoBIOMICS protocol and storre aliquots at -20oC
- 1 vial of DNASE = 2.3ml DNase/RNase free water
- 2 vials of DNASE (for 96 well plate) = 4.6ml DNase/RNase free water. 
SECTION: KingFisher Flex DNA Parallel Protocol
8. Initiate the KingFisher DNA Parallel Protocol on the Kingfisher Flex instrument and load all plates as instructed by the machine. 

BindIT Script for KingFisher Flex
  

PDF From BindIT Software
  
SECTION: KingFisher Flex DNA Parallel Protocol
8.1. After initial binding step, the KingFisher Flex will pause and allow removal of the sample plate. Sample plate with supernatant will be used for subsequent RNA Parallel extraction. 

Seal plate and then either 
- place on ice if extracting RNA the same day as DNA
- place at -20oC (short term) or -80oC (long term) if extracting RNA at a different point. 
SECTION: KingFisher Flex RNA Parallel Protocol
10. Remove sample plate from ice and allow to sit at room temperature for 15 minutes.
Vortex gently after 15 minutes incubation
SECTION: KingFisher Flex RNA Parallel Protocol
11. Transfer 350µl of the supernatant to a new Kingfisher DeepWell plate. 
Add 350µl 95-100% EtoH and 30µl MagBinding Beads 
SECTION: KingFisher Flex DNA Parallel Protocol
8.2. After removal of sample plate, and if running KingFisher RNA Parallel extraction same day, prep all consumables and reagents. 

  
  

SECTION: KingFisher Flex RNA Parallel Protocol
11.1. Original sample plate can be resealed and stored at -80oC to be used for failed RNA extractions (remaining volume should be ~350µl). 
SECTION: KingFisher Flex RNA Parallel Protocol
12. Initiate the KingFisher RNA Parallel Protocol on the Kingfisher Flex instrument and load all plates as instructed by the machine. 

BindIT Script for KingFisher Flex
   

PDF From BindIT Software
  

SECTION: KingFisher Flex RNA Parallel Protocol
12.1. Prep DNase treatment following manufacturere protocol, and keep on ice until KingFisher Flew pauses to allow addition. 
- 1 vial of DNASE = 2.3ml DNase/RNase free water
- 2 vials of DNASE (for 96 well plate) = 4.6ml DNase/RNase free water. 
SECTION: KingFisher Flex RNA Parallel Protocol
13. After Ethanol Wash 2, Kingfisher Flex will pause and allow addition of the prepped DNase plate. Reinitiate Kingfisher RNA Parallel Protocol on addition of DNase plate. 
SECTION: KingFisher Flex RNA Parallel Protocol
14. After DNase digestion, KingFisher will again pause and rotate the sample plate to the entry door to allow manual addition of 500µl of DNA/RNA Prep Buffer to each well. 

After addition, reinitiate KingFisher Flex RNA Parallel Protocol. 
SECTION: KingFisher Flex RNA Parallel Protocol
15. Kingfisher will pause one more time to allow switching of consumable deep well plates with reagents.

Reinitiate the KingFisher RNA Parallel protocol after successful replacement of reagents. 
SECTION: KingFisher Flex RNA Parallel Protocol
16. On completion of KingFisher Flex RNA Parallel protocol, remove the plate with eluted RNA and place on a magnetic stand. Allow to sit for 3 minutes (this is to remove any residual beads which could interfere with down stream protocols). 

On completion of 3 minutes, transfer supernatant to a new 96-well PCR plate. 

Aliquot ~5µl to another 96-well plate for quality control (nanodrop, qubit, tapestation)  and then seal both plates and place at -80oC until ready for downstream processing. 
SECTION: KingFisher Flex DNA Parallel Protocol
9. On completion of KingFisher DNA Parallel, remove plate with eluted DNA and place on a magnetic plate and allow to sit for 3 minutes (this step is to remove any residual beads in eluted DNA which can interfere with downstream protocols)

After 3 minutes transfer supernatant to a new 96-well PCR plate, seal and store at -80oC until ready for downstream processing. 

A good idea is to aliquot ~6µl to another 96 well PCR plate for quality control (nanodrop, qubit, tapestation) thus reducing freeze thaw cycles of DNA.