TITLE: Sanger Tree of Life RNA Extraction: Automated MagMax™ mirVana
AUTHORS: Raquel Juliana Vionette do Amaral, Adam AB Bates, Amy Denton, Halyna Yatsenko, Jessie Jay, Caroline Howard

[DESCRIPTION]
This protocol describes the automated extraction of RNA from multiple different tissue samples intended for RNA-Seq using the MagMax™ mirVana total RNA isolation kit and the Thermo Fisher KingFisher™ Apex. This process is highly effective for the majority of taxonomic groups covered by the Tree of Life Programme, however, challenging samples include corals, jellyfish and annelids. The output of this protocol is a highly concentrated RNA extract which can be diluted and submitted for RNA-Seq on Illumina NovaSeq.

[BEFORE_START]
Add 10 mL of absolute isopropanol to Wash solution 1, mix well and store at room temperature.
Add 48 mL of absolute ethanol to Wash solution 2, mix well and store at room temperature.

[GUIDELINES]
All steps can be performed at room temperature unless stated otherwise.
If samples are not going to proceed to sample lysis immediately, keep samples on dry ice to maintain temperature and prevent nucleic acid degradation.
An experienced operator can expect to comfortably process up to 32 samples, with approximately 2 hours handling time over a start to finish period of 3 hours. This estimation excludes subsequent QC checks.

Additional Notes:
FluidX tubes are used throughout the Tree of Life programme in order to track samples, therefore rather than the microcentrifuge tubes which have been mentioned in this protocol for RNA storage, all routine RNA extracts are stored in FluidX tubes.
Both the KingFisher™ Apex protocol script and the KFX.file have been made available for this protocol - the KFX.file requires ‘BindIx software for KingFisher Apex’ to allow the KingFisher™ Apex protocol to be viewed on a PC or laptop. Alternatively, the file can be transferred directly onto a KingFisher™ Apex instrument using a USB.

[STEPS]
SECTION: Reagent Preparation
1. Prepare the TURBO DNase solution as described below, and once made, store on wet ice: 
  ComponentVolume per sample (µL)MagMAX TURBO DNase Buffer60Turbo DNase (stored in freezer)2.5

SECTION: Reagent Preparation
2. Prepare the Binding Beads Mix as described below, and once made, store on wet ice: 
  ComponentVolume per sample (µL)RNA binding beads12Lysis/binding enhancer (stored in freezer)12

SECTION: Sample Lysis
3. Calculate the amount of lysis buffer required for the samples – 40 µL is required per 1 mg of tissue. 15 mg of sample is used for this protocol, so 600 µL per sample is required.
SECTION: Sample Lysis
4. Create sufficient lysis buffer for your samples:  
  ComponentVolume per sample (µL)Lysis Buffer1000DTT0.7

SECTION: Sample Lysis
5. For samples that require powermashing, transfer 15 mg of tissue into a 1.5 mL BioMasher II tube and add 600 µL of lysis buffer. Disrupt sample in the lysis buffer using a PowerMasher II tissue disruptor and the BioMasher pestle, until no large pieces remain or sample cannot be disrupted further (for more detailed instructions regarding powermashing, please refer to the Sanger Tree of Life Sample Homogenisation: Powermash protocol).
SECTION: Sample Lysis
6. For samples that have been cryoprepped, transfer the 15 mg of cryoprepped tissue into a 2 mL microcentrifuge tube and add 600 µL of lysis buffer. Pipette mix to homogenise the cryoprepped tissue and lysis buffer.
SECTION: Sample Lysis
7. Incubate at room temperature for 30 seconds to 1 minute to allow samples to lyse. If samples will not immediately progress to Step 8, place samples on ice until ready to proceed.
SECTION: Loading and Running the KingFisher™ Apex
8. Transfer 200 µL of each sample directly into individual wells of a Thermo Fisher KingFisher™ 1 mL 96-well deep-well plate.
SECTION: Loading and Running the KingFisher™ Apex
9. Add 100 µL of isopropanol to each sample, seal the plate and shake at room temperature on a plate shaker for 2 minutes at 950 rpm.
SECTION: Loading and Running the KingFisher™ Apex
10. Add 20 µL of the prepared binding beads mix to each sample, re-seal the plate and mix at room temperature on a plate shaker for 5 minutes at 950 rpm.  
 
SECTION: Loading and Running the KingFisher™ Apex
12. On the KingFisher™ Apex, select the protocol (details below in the KingFisher™ Apex RNA Extraction Protocol Script/attached KFX file in the Materials Section) on the protocols list and select using the play button.
SECTION: Loading and Running the KingFisher™ Apex
13. Load the processing plates and the sample plate in the positions prompted by the instrument and then start the run. The full protocol will take approximately 50 minutes.
SECTION: Loading and Running the KingFisher™ Apex
14. After 30 minutes, the protocol will pause and there will be a prompt to remove the DNase plate from the instrument, and add 50 µL of the rebinding buffer and 100 µL absolute isopropanol to each well containing sample as quickly as possible – do not premix these reagents and always add them separately to the wells.
SECTION: Loading and Running the KingFisher™ Apex
15. Load the DNAse plate back into the instrument and press run to resume the protocol.
SECTION: Loading and Running the KingFisher™ Apex
16. At the end of the run, remove the elution plate and store on ice.
SECTION: Loading and Running the KingFisher™ Apex
18. Pipette the eluates into microcentrifuge tubes, perform the required QC, and then store eluates at 
–80°C.
SECTION: Loading and Running the KingFisher™ Apex
11. Prepare the remaining processing plates for the KingFisher™ Apex protocol:
     Plate IDPlate PositionPlate typeReagent(s) requiredVolume per wellSample Plate1Deep-wellSample + isopropanol + binding beads mix200 µL sample + 100 µL isopropanol + 20 µL binding beads mixWash Plate 12Deep-wellWash solution 1150 µLWash Plate 23Deep-wellWash solution 1150 µLDNase Plate4Deep-wellTURBO DNase solution50 µLWash Plate 35Deep-wellWash solution 2150 µLWash Plate 46Deep-wellWash solution 2150 µLElution plate7Standard (200 µL)Elution buffer50 µLTip Comb8Deep-wellPlace a  tip comb in the plate


SECTION: Loading and Running the KingFisher™ Apex
17. Inspect the elution plates for any magnetic beads in the wells. In the rare instance of magnetic beads remaining in the eluate (possible in viscous samples), these samples will need to be transferred to a 1.5 mL microcentrifuge tube and placed on a magnetic rack. Allow around 5 minutes for the beads to migrate and take the clear eluate containing the RNA using a pipette tip.