Saturated phenol

Eight frozen kernels from each ear replicate were pooled and ground in liquid nitrogen with a mortar and pestle. About 100 mg of ground tissue was added to 0.75 ml of saturated phenol, pH 6.6 (Fisher), and homogenized for 2 min. Samples were then dissolved in Tris EDTA buffer, pH 8.0 (ACROS Organics), extracted with 5:1 acid phenol: chloroform, pH 4.5 (Fisher), and precipitated with ice-cold 100% ethanol overnight. Total RNA was further purified with an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. The quality and concentration of RNA was analyzed using an RNA Pico chip on an Agilent Bioanalyzer. The cDNA library construction and sequencing runs on an Illumina HiSeq were done by the Genomic Sciences Laboratory, North Carolina State University. Multiple samples with different barcodes were loaded in three lanes and sequenced to obtain 100 bp single-end reads.

A 200 μL aliquot of an overnight FTG culture of SM101 or its derivative strains was transferred into 10 mL of MDS broth and anaerobically incubated at 37 °C for 3 h. Total RNA was isolated from pelleted cells using the saturated phenol (Fisher Scientific) protocol, as previously described [45 (link)]. Purified RNA was quantified by NanoDrop spectrophotometer (ThermoFisher Scientific) and then stored in a −80 °C freezer. Furthermore, the purity and quality of each extracted RNA sample was evaluated by conducting PCR assay for polC and 16S genes in the absence or presence of reverse transcriptase enzyme. RT-PCR assay was performed using one-step RT-PCR containing 200 ng of purified RNA, avian myeloblastosis virus (AMV) reverse transcriptase (4 U; Promega, Madison, WI, USA), RNA and DNA free dH₂O, 2× DreamTaq Green PCR Master Mix (ThermoFisher Scientific), and appropriate primer sets listed in Table 2. Finally, the RT-PCR assay was carried out using the following parameters: (1) 95 °C for 4 min; (2) 42 °C for 45 min (for cDNA synthesis); (3) 95 °C for 2 min; (4) 30 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and (5) a final extension for 5 min at 72 °C.

A high-loading Rink amide methylbenzhydrylamine (MBHA) resin, Fmoc-L-Cys(Trt)-OH, Fmoc-L-Trp(Boc)-OH, Fmoc-L-Gln(Trt)-OH, Fmoc-L-Tyr(tBu)-OH, Fmoc-L-Leu-OH, Fmoc-L-His(Trt)-OH, Fmoc-L-Phe-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Val-OH, O-(1H-6-Chlorobenzotriazole-1-yl) (HCTU), and N-methyl morpholine (NMM) were purchased from Gyros Protein Technologies, Tucson, AZ, USA. Additionally, N, N-dimethylformamide (DMF), dichloromethane (DCM), acetonitrile, diethyl ether, acetic anhydride, saturated phenol, and ammonium bicarbonate were purchased from Fisher Scientific (Waltham, MA, USA). Piperidine, 2,6-lutidine, trifluoroacetic acid (TFA), and triisopropylsilane (TIPS) were purchased from Sigma-Aldrich (St. Louis, MI, USA).