stock can be stored indefinitely at 20 C and does not need to
be remade with each library construction.
- For First Strand Synthesis, prepare the following mix in a
sterile, RNase-free PCR tube: 500 ng DNA-free RNA, 1μlof
100 μM of your primer mix, and RNase-free water to reach a
total volume of 5μl. Heat at 65C for 5 min and then snap chill
to 4C. At room temperature, prepare a premix (scaled up as
needed) with each sample requiring 5 μl per reaction and
consisting of: 2 μl5 First Strand Buffer, 2 μl10mM
dNTPs, 0.5μl 100 mM dithiothreitol (DTT), and 0.5μl Super-
script III RT. Once prepared, add 5μl premix to 5μl RNA/
primer mix. Heat at 40C for 90 min, cool to 4C (Fig.2a).
- For Second Strand Synthesis, prepare a premix on ice (scaled up
as needed) with each sample requiring 65μl per reaction and
consisting of: 15μl5Second Strand Buffer, 1.5μl10mM
dNTPs, 0.5μl10U/μlE. coliDNA ligase, 2μl10U/μlE. coli
DNA Pol I, 0.5μl2U/μlE. coliRNase H, and 45μlH 2 O.
Once prepared, add 65μl of the ice-cold premix to 10μl of the
First Strand Synthesis reaction. Incubate at 16C for 2 h, and
then add 25μl 0.2 M EDTA (Fig.2b).
- To purify the newly synthesized DNA, use the miniElute PCR
purification kit. This purification step allows buffer exchange,
concentrates the sample, and removes short products and unin-
corporated primers. Add 500μl PB to 100μl Second Strand
Synthesis reaction, apply to the provided column, spin at
17,000g(13,000 rpm in conventional tabletop microcen-
trifuge) for 1 min, and decant the flow-through. Add 750μlPE
to the column, spin again, and decant flow-through. Place
column back in collection tube, and spin again for 2 min to
remove residual PE. Place the column in a fresh collection tube,
open the column’s cap, and air-dry for 2 min with cap open
before elution. Add 12μl EB to the center of the column
membrane, wait 2 min, and spin for 1.5 min to collect eluent.
- End-repair the DNA fragments by using the Quick Blunting
Kit. Prepare a premix on ice (scaled up as needed) with each
sample requiring 3μl per reaction and consisting of: 1.25μl
10 Quick Blunting Buffer, 1.25μl 1 mM dNTPs, and 0.5μl
Blunting Enzyme Mix. Once prepared, add 3μl of the premix
to 10μl of ds-cDNA in a PCR tube. Incubate at 23C for
30 min, 70C for 10 min, and then at 4C (Fig.2c).
- To ligate the adapters use the Quick Ligation Kit. Prepare a
premix on ice (scaled up as needed) with each sample requiring
16.5μl per reaction and consisting of: 15μl2Quick Ligase
Buffer and 1.5μl Quick Ligase. For each sample, add 1μlof
each 12.5μM Fwd (fromstep2) and Rev adapter mix to 13μl
of the end-repaired sample. Add 16.5μl of the Quick Ligation
RNAseq of QS Regulons 185
