premix to the sample/adapter mix, and incubate at 23C for
15 min (seeNote8) (Fig.2d).- To fill-in and PCR amplify each ligation, use Taq DNA Poly-
merase. Prepare a premix on ice (scaled up as needed) with each
sample requiring 90μl per reaction and consisting of: 39μl
H 2 O, 10μl10Reaction Buffer (+Mg), 10μl 25 mM MgCl 2 ,
5 μl dimethyl sulfoxide (DMSO), 10μl10μM Fwd primer,
10 μl10μM Rev primer, 5μl 10 mM dNTPs, and 1μlTaq
DNA Polymerase. Once prepared, add 90μl of premix to 10μl
of ligated sample and subject the mixture to the following
thermal cycling conditions: 72C for 2 min; 95C for 2 min;
20 cycles of (95C for 30 s, 60C for 30 s, 72C for 1 min);
72 C for 5 min; and hold at 4C (Fig.2e). - Purify the DNA by using AMPure XP Magnetic Beads, as bead
purification results in a higher molecular weight cut off than
Qiagen columns. This step will remove any adapter dimer
artifacts that may have been produced during ligation. First,
aliquot 150μl of bead slurry into a clean 1.5 ml microfuge tube
per sample. Allow solution to equilibrate to room temperature
for 20 min. Add 100μl of each PCR reaction to the beads and
mix. Incubate at room temperature for 5 min. Place tubes on
magnet stand for 2 min to capture beads. Remove supernatant
with a pipet and decant. Remove each tube from the stand and
resuspend beads in 200μl 70% EtOH. Capture beads again for
2 min and decant supernatant, as before. Remove each tube
from the stand and wash again with 200μl 70% EtOH. Capture
beads again for 2 min and decant supernatant. To remove the
remaining EtOH, spin briefly (~30 s) in tabletop microfuge at
maximum speed and carefully remove residual EtOH with a
small pipet. Add 20μl of water to the beads and mix well with
the pipette. Let the bead and water mixture sit 1 min, capture
beads on the stand for 2 min, and carefully transfer eluate
containing your RNAseq library to a new labeled tube. - Once the RNAseq library is made, quantify the yield by mea-
suring 2μl of each sample (the total yield should be>1.5μg).
Also, analyze the products on a 2% (wt/vol) agarose gel.
Library fragments should appear as a smear ranging from 120
äFig. 2(continued) Repair using Quick Blunting Kit removes overhangs and adds 5^0 phosphate groups to the
second strand synthesis products. (d) In Adapter Ligation, the stem-loop adapters attach the Illumina P1 and
P2 sequences to the blunt ds-cDNA fragments. The P1/P2 sequences are used as universal amplification sites
for PCR and bridge amplification during cluster generation. When multiplexing is desired, use the barcoded
adapters. (e) The adapter ligation step results in a covalent bond at one end of each insert strand and a nick at
the other end. This nick is repaired with a brief elongation step before PCR to generate a mature sequencing
template for PCR amplification
RNAseq of QS Regulons 187