TITLE: High-Throughput RNA Extraction on Agilent Bravo
AUTHORS: Miriam R Simon, Matt S Zinter, Joseph Derisi

[DESCRIPTION]
Extracting bulk-RNA from biospecimens is a time-consuming process. Column-based extractions performed by hand must be done in batches and the number of samples that can be extracted per batch is dependent on a single person's abilities. Hence, extraction hundreds of samples can take weeks. With every round of extractions, reagents are susceptible to differing amounts of contamination and technical error, resulting in batch effects. Microbial RNA input for metagenomic next-generation sequencing is often found in very low quantities; therefore, contamination introduced during extraction may influence sequencing results and depict an inaccurate reflection of microbes present in the original sample.

Here, we outline a high-throughput, semi-automatic method to extract RNA using a Agilent Bravo liquid handler. This protocol utilizes a magnetic bead-based approach to extracting RNA and is performed in 96-well deep-well plates. All reagents are plated into 96-well deep-well plates before the start of the Bravo protocol. The liquid handler is programmed to aliquot, mix, and aspirate reagents from different decks of plates. The user is only responsible for putting these plates on the correct decks. 

With this approach, we can extract a full plate of 96 samples within a day and reduce the susceptibility to contamination from user error.

[BEFORE_START]
For your sample type, ensure that it is processed/homogenized before plating into sample plate. Inquire with a member of Zinter team for specifics or refer to the Zymo kit manual.

[GUIDELINES]
Bravo protocols are optimized to operate specific depths that are dependent on plate type and manufacturer. For best practice, consistently use the same plates and tips from the same manufacturer to avoid pipetting errors. Even if the plates/tips are thought to be technically identical (based on dimensions), errors can occur.

This Bravo protocol was optimized for nasopharyngeal swabs processed with RNA Shield and bead bashed (before plating).  To extract blood (PAXgene), plasma, BAL, etc., modifications such as centrifuging and/or Proteinase K treatment are required. Inquire with a member of Zinter team for specifics.

The plate shaker is a Bravo attachment that is controlled manually. The shaker in the Derisi Lab is attached to DECK 5.

The time required to pellet Magbinding beads may differ based on the type of magnet used. We use ring magnets to cluster the beads. Optimize before starting. 

[STEPS]
SECTION: Same Day Reagent Preparation
1. Plate and seal all reagents in the biosafety cabinet on the same day you plan to run the procedure with the following volumes:
 
ReagentVolume per wellQuantityPlateLysis Buffer630 uL11 mL deep-wellMagbeads35 uL1Biorad 96-well PCREthanol900 uL11 mL deep-wellEthanol600 uL41 mL deep-wellRNA Wash 1530 uL11 mL deep-wellRNA Wash 2530 uL11 mL deep-wellPrep Buffer530 uL11 mL deep-wellNuclease-free Water50 uL1Biorad 96-well PCRDNase*60 uL1Biorad 96-well PCRInitial Sample**200 uL12 mL deep-wellElution Plate0 uL1Biorad 96-well PCRWaste0 uL32 mL deep-wellWaste0 uL11 mL deep-well
 
SECTION: Bravo Set-Up
2. To begin Bravo set up, switch the instrument on BEFORE opening bravo software (wait about about a minute). NP Swab protocol in DeRisi Lab : GAR_magbead_v6.pro
SECTION: Bravo Set-Up
3. Retrieve 11 tip boxes, spray down Bravo decks and biosafety cabinet first with 70% ethanol, then with RNAse-zap/Windex, and again with ethanol.
SECTION: Bravo Protocol (Pre-Programmed)
4. Place RNA Lysis Buffer on DECK 1, MagBinding beads on DECK 2, Sample plate on DECK 5, Waste (2 mL) on DECK 8, and Tip Boxes on DECKS 3 and 9.
SECTION: Bravo Protocol (Pre-Programmed)
5. Transfer 600 μl RNA Lysis Buffer to Sample plate.
    a. This is done in 4 x 150 μl transfers
    b. Mix and shake Sample plate on DECK 5 for 5 minutes (1/min)
SECTION: Bravo Protocol (Pre-Programmed)
6. Place RNA Lysis Buffer on DECK 1, MagBinding beads on DECK 2, Sample plate on DECK 5, Waste (2 mL) on DECK 8, and Tip Boxes on DECKS 3 and 9.
SECTION: Bravo Protocol (Pre-Programmed)
7. Transfer 600 μl RNA Lysis Buffer to Sample plate. 
a.      This is done in 4 x 150 μl transfers
b.      Mix and shake Sample plate on DECK 5 for 5 minutes
SECTION: Bravo Protocol (Pre-Programmed)
8. Replace RNA Lysis Buffer on DECK 1 with Ethanol (900 ul).
SECTION: Bravo Protocol (Pre-Programmed)
9. Transfer 800 μl Ethanol to the sample and mix well.
a.      This is done in 6 x 133.4 μl transfers.
b.      Mix and shake Sample plate gently on DECK 5 for 5 minutes (half speed).
SECTION: Bravo Protocol (Pre-Programmed)
10. Remove seal from MagBinding Beads plate. Replace tip boxes on DECK 3 and DECK 9 with new boxes.
SECTION: Bravo Protocol (Pre-Programmed)
11. Transfer 30 μl MagBinding Beads to Sample plate.
a. Important: MagBinding Beads settle quickly, ensure that beads are kept in suspension while dispensing. This can be done by adding a mixing step to the MagBinding Beads plate before transferring to the Sample plate.
b. Mix and shake Sample plate gently on DECK 5 for 20 minutes (half speed).
SECTION: Bravo Protocol (Pre-Programmed)
12. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
13. Aspirate and discard the cleared supernatant. 
a.      This is done in 11 x 145.5 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
14. Move Sample plate back to DECK 5. Replace MagBinding Beads with RNA Wash 1 on DECK 2. Replace Waste (2 ml) plate on DECK 8 with an empty Waste (2 ml) plate.
SECTION: Bravo Protocol (Pre-Programmed)
15. Transfer 500 μl RNA Wash 1 to the Sample plate.
a.      This is done in 4 x 125 μl transfers.
b.      Mix and shake Sample plate on DECK 5 for 5 minutes
SECTION: Bravo Protocol (Pre-Programmed)
16. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
17. Aspirate and discard the cleared supernatant.
a.      This is done in 4 x 125 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
18. Move Sample plate back to DECK 5. Replace RNA Wash 1 with RNA Wash 2 on DECK 2. Replace tip boxes on DECK 3 and DECK 9 with new boxes.
SECTION: Bravo Protocol (Pre-Programmed)
19. Transfer 500 μl RNA Wash 2 to the Sample plate.
a.      This is done in 4 x 125 μl transfers.
b.      Mix and shake Sample plate on DECK 5 for 5 minutes
SECTION: Bravo Protocol (Pre-Programmed)
20. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
21. Aspirate and discard the cleared supernatant.
a.      This is done in 4 x 125 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
22. Move Sample plate back to DECK 5. Replace Ethanol (900 ul) with Ethanol 1/4 on DECK 1.
SECTION: Bravo Protocol (Pre-Programmed)
23. Transfer 500 μl Ethanol 1/4 to the Sample plate.
a.      This is done in 4 x 125 μl transfers.
b.      Mix and shake Sample plate on DECK 5 for 5 minutes
SECTION: Bravo Protocol (Pre-Programmed)
24. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
25. Aspirate and discard the cleared supernatant.
a.      This is done in 4 x 125 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
26. Move Sample plate back to DECK 5. Replace Waste (2 ml) plate on DECK 8 with an empty Waste (2 ml) plate. Replace Ethanol 1/4 with Ethanol 2/4 on DECK 1. Replace tip boxes on DECK 3 and DECK 9 with new boxes.
SECTION: Bravo Protocol (Pre-Programmed)
27. Transfer 500 μl Ethanol 2/4 to the Sample plate. 
a.      This is done in 4 x 125 μl transfers.
b.      Mix and shake Sample plate on DECK 5 for 5 minutes
SECTION: Bravo Protocol (Pre-Programmed)
28. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
29. Aspirate and discard the cleared supernatant.
a.      This is done in 4 x 125 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
30. Move Sample plate back to DECK 5. Replace RNA Wash 2 with DNase plate on DECK 2 and confirm it is unsealed. Replace tip boxes on DECK 3 and DECK 9 with new boxes.
SECTION: Bravo Protocol (Pre-Programmed)
31. Transfer 50 μl DNase treatment to the Sample platea. Mix and shake Sample plate gently on DECK 5 for 10 minutes (half speed).
SECTION: Bravo Protocol (Pre-Programmed)
32. Replace DNase plate on DECK 2 with Prep Buffer plate.
SECTION: Bravo Protocol (Pre-Programmed)
34. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
35. Aspirate and discard the cleared supernatant.
a.      This is done in 4 x 137.5 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
36. Move Sample plate back to DECK 5. Replace tip boxes on DECK 3 and DECK 9 with new boxes. Replace Ethanol 2/4 with Ethanol 3/4 on DECK 1
SECTION: Bravo Protocol (Pre-Programmed)
37. Transfer 500 μl Ethanol 3/4 to the Sample plate.
a.      This is done in 4 x 125 μl transfers.
b.      Mix and shake Sample plate on DECK 5 for 5 minutes
SECTION: Bravo Protocol (Pre-Programmed)
38. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
39. Aspirate and discard the cleared supernatant.
a.      This is done in 4 x 125 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
40. Move Sample plate back to DECK 5. Replace Ethanol 3/4 with Ethanol 4/4 on DECK 1. Replace Waste (2 ml) plate on DECK 8 with an empty Waste (2 ml) plate.
SECTION: Bravo Protocol (Pre-Programmed)
41. Transfer 500 μl Ethanol 4/4 to the Sample plate.
a.      This is done in 4 x 125 μl transfers.
b.      Mix and shake Sample plate on DECK 5 for 5 minutes
SECTION: Bravo Protocol (Pre-Programmed)
42. Move the Sample plate/tube to the magnetic stand at DECK 7. Incubate for 4 minutes until beads have pelleted.
SECTION: Bravo Protocol (Pre-Programmed)
43. Aspirate and discard the cleared supernatant.
a.      This is done in 4 x 125 μl transfers.
SECTION: Bravo Protocol (Pre-Programmed)
44. Replace Prep Buffer with Water plate on DECK 2. Dry the beads for 30 minutes or until dry.
SECTION: Bravo Protocol (Pre-Programmed)
45. Confirm Water plate is unsealed. Place Elution PCR plate on DECK 4. Move Sample plate back to DECK 5. Replace tip boxes on DECK 3 and DECK 9 with new boxes.
SECTION: Bravo Protocol (Pre-Programmed)
46. Transfer 45 μl of Water to the Sample plate on DECK 5.
a.      Mix and shake Sample plate on DECK 5 for 10 minutes
SECTION: Bravo Protocol (Pre-Programmed)
47. Move sample plate to DECK 7. Incubate for 4 minutes.
SECTION: Bravo Protocol (Pre-Programmed)
48. Transfer 40 μl from the Sample plate to Elution plate on DECK 4. Freeze Elution plate and Sample plate in -80°C and discard all plates and tip boxes.
SECTION: Bravo Protocol (Pre-Programmed)
33. 28.   Transfer 500 μl Prep Buffer to Sample plate.
a.      This is done in 4 x 125 μl transfers.
b.      Mix and shake Sample plate on DECK 5 for 10 minutes.