Taq master mix

Open complex formation was performed as previously described (Hahn and Roberts, 2000 (link); Sasse-Dwight and Gralla, 1991 (link)) with a few minor modifications. Transcription reactions were formed as described for the in vitro transcription assays except 15 µg of indicated extract and 50 ng of rDNA reporter plasmid template was used and DTT and RNase inhibitor were omitted. After 30 min at room temperature, KMnO₄ was added to a final concentration of 10 mM and incubated for 2 min and then stopped by 3 µl of 2-mercaptoethanol followed by 180 µl of transcription stop mix. Reactions were extracted with phenol/chloroform and ethanol precipitated. Modified DNAs were resuspended in water and were used as templates for primer extension with Taq master mix (New England Biolabs, Ipswich, MA) using a LacI primer labeled with Cyanine 5.5 on the 5’ end. The following thermocycler conditions were used: 95°C for 2 min, then 18 cycles of 95°C for 30 s, 55°C for 30 s, and 68°C for 1 min, followed by 5 min at 68°C. Reactions were analyzed on a 7% Urea-PAGE gel and quantitated by Odyssey FC imager (LiCOR, Lincoln, NE) using the 700 nm wavelength channel. Recovery of open complex activity for mutant extracts was performed by pre-incubating the indicated extracts with 20 ng of recombinant Core Factor (rCF) for 20 min on ice with intermittent mixing prior to adding to the transcription reactions.

The wild-type parent ATCC 3624 and derivatives (the agrB-null mutant or complementing strain) were grown in TY medium for 5 h at 37°C. Cultures were then pelleted and RNA was extracted using saturated phenol and purified by TRIzol and chloroform (Life Technology and Sigma), as previously described (36 (link)). After the absence of DNA was confirmed (36 (link)) by subjecting samples to PCR without reverse transcriptase, RNA was quantified by measuring the sample absorbance at 260 nm. An aliquot of 1 μl of purified RNA (100 ng) was then used in a one-step RT-PCR containing 10 μl of 2× Taq master mix (New England Biolabs), 4 U of avian myeloblastosis virus reverse transcriptase (Promega), and agrB gene primers (described earlier), with ddH₂O added to reach a 20-μl total volume. Similarly, 16S RNA RT-PCR was performed as a loading control (36 (link)). Reaction mixtures were incubated for 45 min at 45°C to allow cDNA synthesis, and then regular PCR cycling was performed using the following conditions: (i) 95°C for 2 min; (ii) 30 cycles of 95°C for 15s, 50°C for 30 s, and 68°C for 30 s; and (iii) a final extension of 68°C for 5 min.

To determine the best restriction enzyme (RE) system for reducing the complexity of the fox genome, DNA (100 ng) from a single individual was digested, separately, with several REs (ApeKI, EcoT22I, PstI, and EcoT22I/PstI double digest) according to the enzyme manufacturer’s protocol (New England Biolabs, Ipswich, MA). Samples were digested at 37°C for 2 hours and then, incubated at 80°C for 20 minutes to inactivate REs. Digested DNAs were ligated to adapters as previously described [22 ]. Samples were then purified (QIAquick PCR Purification Kit; Qiagen, Valencia, CA) and 2 μl of each library was amplified in a 50 μl volume containing 1 x Taq master mix (New England Biolabs, Ipswich, MA) and 25 pmol of each of two primers containing complementary sequences to ligated adapters and Illumina solid-phase oligonucleotides bound to the flowcell lane surface. PCR was performed following the previously described protocol [22 ]. Amplified libraries were purified again, as above, and fragments were visualized using the Experion (Bio-Rad, Hercules, CA) (S1 Fig). DNA profiles for ApeKI and EcoT22I enzymes showed enrichment for fragments of desirable sizes (S1 Fig) and did not contain repeat-associated peaks, therefore libraries were made with both of these enzymes.

RNA was isolated as previously described (34 (link)). Briefly, for expression screening, wild-type SM101 was grown in MDS medium for 2 or 4 h or TGY media for 2, 4, or 8 h at 37°C. For confirming insertional mutagenesis by the TargeTron system, wild-type SM101, SM101-CPR0195KO, SM101-CPR0195comp, and SM101-CPR1055KO strains were grown in MDS medium for 2 h. Cultures were then pelleted at the indicated times, and RNA was extracted using saturated phenol. After extraction with phenol, the RNA-containing aqueous phase was twice extracted using TRIzol and chloroform. Following treatment with DNase to remove any residual DNA, RNA was quantified by determining the absorbance at 260 nm. Next, 1 µl of purified RNA was used in a one-step RT-PCR containing 2× Taq Master Mix (New England Biolabs), MilliQ water, and primers specific for the cpr0195 gene, cpr1055 gene, or polC gene (Table 1). Duplicate reaction mixtures were set up, one containing avian myeloblastosis virus (AMV) reverse transcriptase (4 U; Promega) and the other without AMV reverse transcriptase as a control. Reaction mixtures were incubated for 45 min at 37°C to allow cDNA synthesis, before PCR cycling as follows: (i) 95°C for 4 min; (ii) 35 cycles of 94°C for 30 s, 50°C for 30 s, and 68°C for 30 s; and (iii) a final extension of 68°C for 10 min.

The two MUC1 genotype polymorphisms (rs4072037 and rs2070803) were determined by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method with PCR primer pairs listed in Table 1.
The PCR was carried out in a 30 μl reaction mix containing 100 ng of DNA template, 1 unit of Taq Mastermix (New England BioLabs, Beverly, MA, USA), 0.5 μM of forward primer, and 0.5 μM of reverse primer. The PCR was carried out in a thermocycler, and reaction conditions consisted of 95°C denaturation for 5 min, 94°C annealing for 30 s, 62°C annealing for 30 s, 72°C annealing for 30 s (40 cycles), and 72°C elongation for 10 min. The PCR products were digested with appropriate restriction enzymes (New England BioLabs, Beverly, MA, USA) at 37°C for 8 h. The restriction enzymes were AlwNI (rs4072037 G/A) and TaqaI (rs2070803 G/A). The PCR and digestion products were analyzed with a 1.5% agarose (Serva, Germany) gel with intercalating dye (ethidium bromide) staining. Selected PCR-amplified DNA samples (about 5%) were analyzed by DNA sequencing (Figure 1).

Young leaves were harvested from 242 plants and DNA was extracted using the Qiagen DNeasy 96 Plant Kit (Qiagen, Valencia, CA, USA) following the manufacturer’s instructions. A NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Inc. Wilmington, DE, USA) was used for measuring DNA concentration and quality. The extracted DNA was submitted to the University of Minnesota Genomics Center for processing and sequencing using the GBS protocol according to Elshire et al. [67 (link)]. Briefly, genomic DNA (100 ng) was digested with 10 units of ApeKI (NEB) and incubated at 75 °C for 2 h. Phased adaptors with a three-base overhang on the 5′ ends of the bottom strand were ligated with digested DNA and 200 units of T4 ligase (NEB) at 22 °C for 1 h and heat-inactivated. The ligated samples were purified, and bar codes were added by 18 cycles with 2X NEB Taq Master Mix. Lastly, pooled libraries were size-selected for the 300 to 744 bp library region (156 to 600 DNA inserts). The final pool was then diluted to 1 nM and sequenced on the Illumina NovaSeq 6000 using single-end 1 × 100 reads on a single lane of an SP variant flowcell.