RNA (and optional DNA) extraction from environmental samples (filters)
Take frozen tubes out of the -80C freezer, keep on ice.
While filtere are thawing, add RNase/DNase-free 0.5mm Silica beads to each sample tubea.
If RLT+ buffer (with beta mercaptoethanol) was not added previously, add it here.
Vortex for 5 minutes to ensure beads disrupt filter
a. Ensure filter remains in RLT+ buffer the whole time
b. Options to increase yield:
i. Add pre-heated RLT+ buffer (heat for 2-3 minutes at 65C)
ii. Place tube on Tissue lyser for bead beating
iii. Vortex for additional minutes Once thoroughly lysed (note foam/bubbles may have appeared), transfer liquid lysate to new tube (avoid transferring filter).
Using sterile forceps, transfer the filter carefully into a 5mL sterile syringe.
Squeeze out excess lysate from filter through the syringe into the new tube of lysate.
New tube for each sample should only contain lysate.
a. Optional step is to run this lysate through a Qiashredder for additional lysis.
**Start with Qiagen extraction steps here (following Qiagen DNA/RNA All prep kit): 
Transfer the lysate to the AllPrep DNA spin column.
Centrifuge for 30 seconds at > 10,000 rpm.
Transfer flow-through (filtrate) into a new tube for RNA purification.
This DNA column can now be stored in the fridge until DNA extraction (4C).
a. If multiple spins of the lysate are required, continue this until all lysate has been passed through the DNA column and all flow-through has been obtained in a new tube.
Add 1 volume of 70% EtOH to the RNA flow-through product, mix by pipetting.
Do not centrifuge.
Add 1 volume of 70% EtOH to the RNA flow-through product, mix by pipetting.
Do not centrifuge.
Transfer up to 700µl of sample (including any precipitate) to an RNeasy spin column.
Centrifuge for 30 seconds at > 10,000 rpm.
Discard the flow-through.
Add 350µl of Buffer RW1 to RNeasy spin column.
Centrifuge for 15 seconds at > 10,000 rpm.
Discard the flow-through.
Make up DNase I and Buffer RDD stock mix for DNase digestion.
For each sample add 10µl of DNaseI stock to 70µl of Buffer RDD, mix solution and centrifuge briefly.
Add DNaseI mix (80µl) directly to RNase spin column.
Incubate at room temperature for 15 minutes.
Again, add 350µl of Buffer RW1 to RNeasy spin column.
Centrifuge for 15 seconds at > 10,000 rpm.
Discard the flow-through.
Add 500µl of Buffer RPE to RNeasy spin column.
Centrifuge for 15 seconds at > 10,000 rpm.
Discard the flow-through.
Again, add 500µl of Buffer RPE to RNeasy spin column.
Centrifuge for 2 minutes at > 10,000 rpm.
Discard the flow-through.
Option to place RNeasy spin column into a new 2mL collection tube and centrifuge at full speed for 1 minute – this will eliminate any possible carry over of Buffer RPE.
Place RNeasy spin column into a new 1.5ml collection tube, add 30-50µl RNase-free water.
Centrifuge for 1 minute at > 10,000 rpm to elute the RNA.
Options to increase yield:
i. Pre-heat RNase-free water ahead of addition to RNeasy column
ii. Let RNase free water sit on RNeasy column for 1-2 minutes before centrifugation
iii. Transfer eluted RNA back into the RNeasy column and re-centrifuge to increase concentration.
Genomic DNA purification Grab the DNA spin columns out of the fridge.
Add 500µl Buffer AW1 to All Prep DNA spin column.
Centrifuge for 15 seconds at > 10,000 rpm.
Discard the flow-through.
Again add 500µl of Buffer AW2 to the DNA column.
Centrifuge for 2 minutes at full speed.
Discard the flow-through.
Place DNA column into a new 1.5ml collection tube, add 50-100µl of Buffer EB to the column.
Let sit at room temperature on the column for 1 minute.
Centrifuge for 1 minute at > 10,000 rpm.
Options to increase yield:
i. Pre-heat EB buffer water ahead of addition to DNA column
ii. Transfer eluted DNA back into the RNeasy column and re-centrifuge to increase concentration.
QC both DNA and RNA product using both Qubit fluorometer (concentration) and Agilent Bioanalyzer (quality).
a. For the Qubit load 2µl of product to the 198µl of buffer/dye solution for each sample
