TITLE: RNA and DNA extraction (Qiagen) of frozen tissue to nanopore sequencing 
AUTHORS: Eric Pederson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RNA extraction of extraction using the Qiagen Allprep RNA extraction kit followed by nanopore sequencing on the MinION.</div></div>

[STEPS]
?. [Pre-experiment]
Book RNA extraction room
?. [Pre-experiment]
Spray  with ETOH and then  in the fume hood
?. [Pre-experiment]
Prepare labelled 2 ml low bind tubes with 4  Zirconium Oxide Beads 2.0 mm RNase Free 4 mL beads in each
?. [Pre-experiment]
Make 3 1.5 ml eppendorf tubes for each sample; 1 with sterile water, 1 with 99% ETOH and 1 with
?. [Pre-experiment]
From the Add 1 ml buffer RLT to 10 ul b-mercaptoethanolEach sample requires 600 ul of buffer RLT + b-mercaptoethanolSo for example for 6 samples one would need 3.6 ml of the buffer RLT + b-mercaptoethanol mix. Thus, one would need to prepare 4 ml buffer RLT + 40 ul b-mercaptoethanol.
?. [Tissue destruction]
Add to the 2.0 ml eppidendorf tube that already has the beads (Zirconium Oxide Beads 2.0 mm RNase Free 4 mL) with 600 ul RLT buffer and b-mercaptoethanol mixture.
?. [Tissue destruction]
In the most sterile way possible, cut a small piece of tissue, half a pea size or so, and put it into one of the labelled 2ml tubes with Beads and the RLT mixture on iceMake sure to use the ethanol to clean everything and not to cut yourself.Use the forceps to grab the tissue that flies away.
?. [Tissue destruction]
Use the bullet blender to destroy the tissue (Tape on lids)- 3 minutes - speed setting 8
?. [Tissue destruction]
Get tissue from the -80 freezer and place on dry ice
?. [Tissue destruction]
Materials to take to the basement -80° freezer;- gloves- white warm gloves- dry ice- blades- wipes- forceps- ethanol spray- petri-dishes (tissue type written on the lid)- marker- paper tape- normal ice- tubes with Beads, PBS and  on ice
?. [Tissue destruction]
Centrifuge the lysate for 3 min at maximum speed.
?. [Pre-experiment]
Label all the spin columns and the collection tubes properly
?. [Tissue destruction]
From the Carefully remove the supernatant by pipetting, and transfer it to the AllPrep DNA spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 30 s at ≥ 8000 x g (≥10,000 rpm).
?. [Tissue destruction]
Place the AllPrep DNA spin column in a new 2 ml collection tube (supplied), and store at room temperature (15–25oC) or at 4oC for later DNA purification in steps 14–17. Use the flow-through for RNA purification in the following steps
?. [RNA extraction]
Add 1 volume (usually 600 μl) of 70% ethanol to the flow- through from step 5, and mix well by pipetting. Do not centrifuge.
?. [RNA extraction]
Transfer up to 700 μl of the sample, including any precipitate that may have formed, to an RNeasy spin column placed in a 2 ml collection tube (supplied). Close the lid gently, and centrifuge for 15 s at ≥ 8000 x g (≥ 10,000 rpm). Discard the flowthrough.
?. [RNA extraction]
Add 350 μl Buffer RW1 to the RNeasy spin column, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through.* Reuse the collection tube in step E4.
?. [RNA extraction]
Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 15 s at ≥ 8000 x g (≥ 10,000 rpm) to wash the spin column membrane. Discard the flow-through.
?. [RNA extraction]
Add 500 μl Buffer RPE to the RNeasy spin column. Close the lid gently, and centrifuge for 2 min at ≥ 8000 x g (≥ 10,000 rpm) to wash the spin column membrane
?. [RNA extraction]
Place the RNeasy spin column in a new 2 ml collection tube (supplied), and discard the old collection tube with the flow- through. Centrifuge at full speed for 1 min.
?. [RNA extraction]
Place the RNeasy spin column in a new 1.5 ml collection tube (supplied). Add 30 μl RNase-free water directly to the spin column membrane. Close the lid gently, and centrifuge for 1 min at ≥ 8000 x g (≥10,000 rpm) to elute the RNA.
?. [RNA extraction]
If the expected RNA yield is > 30 μg, repeat the previous step using another 30 μl of RNase-free water, or using the eluate from step 12 (if high RNA concentration is required). Reuse the collection tube from the previous step.
?. [RNA extraction]
Run the tapestation and the nanodrop on the RNA.
?. [RNA extraction]
Store the RNA in the -80 freezer for long term storage, or leave on ice until after the DNA extractions to preform the cDNA synthesis
?. [DNA extraction]
Add 500 μl Buffer AW1 to the AllPrep DNA spin column from before the RNA extraction. Close the lid gently, and centrifuge for 15 s at ≥ 8000 x g (10,000 rpm). Discard the flow-through.
?. [DNA extraction]
Add 500 μl Buffer AW2 to the AllPrep DNA spin column. Close the lid gently, and centrifuge for 2 min at full speed to wash the spin column membrane.
?. [Pre-experiment]
Buffer AW1 is supplied as a concentrate. Ensure that ethanol is added to Buffer AW1 before use Buffer AW2 is supplied as a concentrate. Ensure that ethanol is added to Buffer AW2 before use
?. [DNA extraction]
Place the AllPrep DNA spin column in a new 1.5 ml collection tube (supplied). Add 100 μl Buffer EB directly to the spin column membrane and close the lid. Incubate at room temperature (15–25oC) for 1 min, and then centrifuge for 1 min at ≥ 8000 x g (10,000 rpm) to elute the DNA.
?. [DNA extraction]
Repeat previous step to elute further DNA.
?. [DNA extraction]
Run the tapestation (genomic) and the Qubit on the DNA.
?. [DNA extraction]
Store the DNA in the -20° or -80° freezer for long term storage.
?. [cDNA synthesis]
From the Prepare the following reaction in a 0.2 ml PCR tube
?. [cDNA synthesis]
Mix gently by flicking the tube and spin down
?. [cDNA synthesis]
(X ul) 1 ng PolyA+ RNA (or ~50 ng total RNA)(1 ul) VN primers (VNP), at 2 uM(1 ul) 10 mM dNTPs(9-X ul) RNase-free waterTotal volume of 11 ul
?. [cDNA synthesis]
Incubate at 65° for 5 minutes and then snap cool on a pre-chilled freezer block
?. [cDNA synthesis]
In a separate 0.2 ml PCR tube, mix the following reaction
?. [cDNA synthesis]
Mix gently by flicking the tube and spin down
?. [cDNA synthesis]
(4 ul) 5x RT buffer(1 ul) RNase Out(1 ul) Nuclease-free water(2 ul) Strand-Switching Primer (SSP, at 10 uM)Total vollume of 8 ul
?. [cDNA synthesis]
Add the strand-switching buffer to the snap-cooled, annealed mRNA and mix by flicking the tube and spin down
?. [cDNA synthesis]
Incubate at 42° for 2 minutes
?. [cDNA synthesis]
Add 1 ul of Maxima  H minus Reverse Transciptase.The total volume should now be 20 ul
?. [cDNA synthesis]
Mix by flicking the tube and spin down
?. [cDNA synthesis]
Incubate using the following protocol
?. [Adapator extension]
Serapure/Ampure purification
?. [Adapator extension]
Add adaptors to the cDNA using PCR
?. [cDNA synthesis]
ABCD1Cycle stepTempTime# of cycles2Reverse Transcription and strand-switching42°90 minutes13Heat inactivation85°5 minutes14Hold4°∞-
ABCD1Cycle stepTempTime# of cycles2Reverse Transcription and strand-switching42°90 minutes13Heat inactivation85°5 minutes14Hold4°∞-
?. [Adapator extension]
From the And Reaction mix;(10 ul) 5x Prime STAR GXL buffer(1 ul) 10 mM KAPA dNTP mix(1 ul) Prime STAR GXL DNA polymerase(5 ul) cDNA(1 ul) primer (Barcode mix from nanopore
?. [Adapator extension]
PCR program1 cycle98° - 30 seconds10 cycles98° - 10 seconds64° - 15 seconds68° - 10 seconds1 cycle68° - 5 minutes4° - ∞
?. [Ligation reaction]
First step
?. [Adapator extension]
Bead extraction:- Add 1.8X beads to samples  - flick to mix- incubate for 10 minutes at room temperature- use the magnet to collect the DNA-beads- discard supernatant- wash with 70% ethanol- dry at 37° for 3 minutes- elute with 20 ul TE buffer- incubate for 5 minutes at room temperature- use the magnet to collect the DNA-beads- collect supernatant into a new tube
?. [Ligation reaction]
Ligation reaction
?. [Ligation reaction]
Incubate for 10 minutes at room temperature
?. [Ligation reaction]
From the (7 ul) Ultra II End prep  reaction buffer (NEB, E7647AA)(3 ul) Ultra I End prep Enzyme mix (NEB, E7646AA)(50 ul) cDNA with adaptersTotal volume of 60 ul
?. [Ligation reaction]
From the (60 ul) purified DNA(25 ul) Ligation buffer(10 ul) Ligase(5 ul) adaptor mix
?. [Ligation reaction]
Run Qubit
?. [Loading Nanopore flow cell]
From theAnd using a DNA library - 12 ul ( of the 14 ul)Sequencing buffer (SQB) - 37.5 ulLoading Beads - 25.5 ulTotal volume of 75 ul
?. [Ligation reaction]
PCR program20° for 5 minutes65° for 5 minutes
?. [Ligation reaction]
Serapure/Ampure purification using a 1:1 mix ration (60:60)
?. [Ligation reaction]
Bead extraction:- Add 1.8X beads to samples  - flick to mix- incubate for 10 minutes at room temperature- use the magnet to collect the DNA-beads- discard supernatant- wash with 70% ethanol- dry at 37° for 3 minutes- elute with 20 ul TE buffer- incubate for 5 minutes at room temperature- use the magnet to collect the DNA-beads- collect supernatant into a new tube
?. [Loading Nanopore flow cell]
Thaw the Sequencing Buffer (SQB), Loading Beads (LB), Flush Tether (FLT)  and one tube of Flush Buffer (FB) at RT before placing the tubes on ice as soon as thawing is complete
?. [Ligation reaction]
Modified Serapure/Ampure bead purification
?. [Ligation reaction]
- Add 1.8X beads to samples  - flick to mix- incubate for 10 minutes at room temperature- use the magnet to collect the DNA-beads- discard supernatant- wash with SFB (250 ul)- dry at 37° for 3 minutes- elute with 15 ul TE buffer- incubate for 5 minutes at room temperature- use the magnet to collect the DNA-beads- collect supernatant into a new tube
?. [Pre-experiment]
From the Make DNase solution.
?. [Pre-experiment]
Prepare DNase I stock solution before using the RNase-Free DNase Set for the first time. Dissolve the solid DNase I (1500 Kunitz units) in 550 μl of the RNase-free water provided. To avoid loss of DNase I, do not open the vial. Inject RNase-free water into the vial using an RNase-free needle and syringe. Mix gently by inverting the vial. Do not vortex.
?. [Pre-experiment]
For long-term storage of DNase I, remove the stock solution from the glass vial, divide it into single-use aliquots, and store at –20oC for up to 9 months. Thawed aliquots can be stored at 2–8oC for up to 6 weeks. Do not refreeze the aliquots after thawing.
?. [RNA extraction]
Add 10 μl DNase I stock solution (see above) to 70 μl Buffer RDD. Mix by gently inverting the tube, and centrifuge briefly to collect residual liquid from the sides of the tube. Buffer RDD is supplied with the RNase-Free DNase Set. Note: DNase I is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube. Do not vortex.
?. [RNA extraction]
Add the DNase I incubation mix (80 μl) directly to the RNeasy spin column membrane, and incubate at room temperature (20–30oC) for 15 min. Note: Be sure to add the DNase I incubation mix directly to the RNeasy spin column membrane. DNase digestion will be incomplete if part of the mix sticks to the walls or the O-ring of the spin column.
?. [RNA extraction]
Add 350 μl Buffer RW1 to the RNeasy spin column, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through.Continue with step 9 (22) of the protocol on page 26 (i.e., the first wash with Buffer RPE). Reuse the collection tube in step 9 (22).
?. [Pre-experiment]
Turn on centrfuge to 4°.
?. [Loading Nanopore flow cell]
Mix the Sequencing Buffer (SQB) and Flush Buffer (FB) tubes by vortexing, spin down and return to ice.
?. [Loading Nanopore flow cell]
Spin down the Flush Tether (FLT) tube, mix by pipetting, and return to ice.
?. [Loading Nanopore flow cell]
Open the lid of the nanopore sequencing device and slide the flow cell’s  priming port cover clockwise so that the priming port is visible.
?. [Loading Nanopore flow cell]
After opening the priming port, check for small bubbles under the cover.  Draw back a small volume to remove any bubble (a few µl) as follows:
?. [Loading Nanopore flow cell]
Prepare the flow cell priming mix as follows:
?. [Loading Nanopore flow cell]
Set a P1000 pipette to 200 µl
?. [Loading Nanopore flow cell]
Turn the wheel until the dial shows 220-230 µl, or until you can see a small volume of buffer entering the pipette tipCare must be taken when drawing back buffer from the flow cell. The  array of pores must be covered by buffer at all times. Removing more  than 20-30 µl risks a loss of sequencing channels.
?. [Loading Nanopore flow cell]
Insert the tip into the priming port
?. [Loading Nanopore flow cell]
Care must be taken when drawing back buffer from the flow cell. The  array of pores must be covered by buffer at all times. Removing more  than 20-30 µl risks a loss of sequencing channels.
?. [Loading Nanopore flow cell]
Add 30 µl of thawed and mixed Flush Tether (FLT) directly to the tube  of thawed and mixed Flush Buffer (FB), and mix by pipetting up and  down.
?. [Loading Nanopore flow cell]
Load 800 µl of the priming mix into the flow cell via the priming port,  avoiding the introduction of air bubbles. Wait for 5 minutes.
?. [Loading Nanopore flow cell]
Thoroughly mix the contents of the Loading Beads (LB) by pipetting. Note  that The Loading Beads (LB) tube contains a suspension of beads. These beads settle very quickly. It is vital that they are mixed immediately before use.
?. [Loading Nanopore flow cell]
In a new tube, prepare the library for loading as follows:
?. [Loading Nanopore flow cell]
37.5 µl Sequencing Buffer (SQB)25.5 µl Loading Beads (LB), mixed immediately before use12 µl cDNA library prepared as described in SOP ID:0005
 75 ul Total
?. [Loading Nanopore flow cell]
Complete the flow cell priming:
?. [Loading Nanopore flow cell]
Gently lift the SpotON sample port cover to make the SpotON sample port accessible.Load 200 µl of the priming mix into the flow cell via the priming  port (not the SpotON sample port), avoiding the introduction of air  bubbles.
?. [Loading Nanopore flow cell]
Mix the prepared library gently by pipetting up and down just prior to loading.
?. [Loading Nanopore flow cell]
Add 75 μl of sample to the flow cell via the SpotON sample port in a  dropwise fashion. Ensure each drop flows into the port before adding the  next.
?. [Loading Nanopore flow cell]
Gently replace the SpotON sample port cover, making sure the bung enters  the SpotON port, close the priming port and replace the MinION lid.