Quorum Sensing

10. Digest away the DNA in the RNA sample by mixing in a tube
    on ice: 10μg RNA, 10μl10
    TURBO DNase Buffer, 2μl
    TURBO DNase, and RNase-free water to bring the total vol-
    ume up to 100μl. Incubate the tube in a 37C water bath for
    30 min. Give the tube a quick spin to collect condensation, and
    add another 2μl of TURBO DNase, mix by inverting tube,
    give it another quick spin, and incubate the tube in 37C water
    bath for another 30 min.


11. To clean the RNA again, pipet 350μl Buffer RLT (+1:100
    βME) into the DNase treated tube (total volume now at
    450 μl) and mix thoroughly by pipetting. Add 675μlof100%
    ethanol to the sample and mix thoroughly by pipetting. Apply
    700 μl of the sample to the RNeasy MinElute column (columns
    arestoredat4Cuponarrival), centrifugefor15sat14,000rpm
    (17,000
    g), and discard the flow-through. Repeat using the
    remainder of the sample in 700μl increments. Transfer the
    RNeasy MinElute column into new 2 ml collection tube. Add
    500 μl Buffer RPE to the RNeasy MinElute column, centrifuge
    for 15 s at 14,000 rpm (17,000
    g), and discard the flow-
    through. Add 500μl of 80% ethanol to the RNeasy MinElute
    column and centrifuge for 2 min at 14,000 rpm (17,000
    g).
    Transfer the RNeasy MinElute column into new 2 ml microfuge
    tube, open the lid of the RNeasy MinElute column, and centri-
    fuge for 5 min at 14,000 rpm (17,000
    g) (don’t forget to
    label the tube body!). Transfer the RNeasy MinElute column
    into new 1.5 ml tube and open the lid of the RNeasy MinElute
    column for 2.5 min to remove any residual ethanol. Pipet
    12–20μl RNase-free water to obtain 1 μg/μl RNA directly
    onto the MinElute column membrane, let the column stand for
    1 min, and centrifuge for 1 min at17,000
    g. Immediately
    place RNA on ice and store at 80 C.


12. Again, determine the RNA concentration, run the RNA on an
    agarose gel for quality (seesteps8and 9 ), and verify that the
    RNA is DNA-free by using PCR. Set up a PCR reaction with a
    primer set to a region in the genome of your organism. Use the
    appropriate PCR conditions for your primer set, use 500 ng
    RNA, and perform the PCR amplification step for 30–35
    cycles. Take care to use a positive control (pure gDNA) and a
    negative control (no template). Run each sample on an agarose
    gel to confirm that the RNA sample and the negative control
    have no DNA (no PCR product), and that the positive control
    contains the desired DNA PCR product (seeNote6).

3.2 RNAseq Library
Construction (See
Fig.2 for Overview)

1. Obtain a primer set for your organism that will limit amplifica-
   tion of ribosomal RNA (seeNote7).


2. To generate the barcoded adapters, combine 12.5μl of 100μM
   RevAD with 12.5μl of 100μM Fwd Adapter in a 1.5 ml
   microfuge tube. Add 75μl of water and mix well. This working

184 Charlotte D. Majerczyk