Nanodrop nd 1000 system

RNA extraction was carried out using the Zymo Direct-zol RNA MiniPrep kit (R2071, Zymo Research, Irvine, CA, USA) and applying few modifications to the manufacturer’s instructions. Briefly, after adding up to 700 µL of the Tri reagent, the mechanical lysis of cells was achieved using a Vortex Genie 2 instrument (Mo Bio Laboratories Carlsbad, CA, USA) by performing two rounds of 20 min at the highest speed alternating with 3 min on ice. The quantity of total RNA was measured spectrophotometrically using a Nanodrop Nd 1000 system (Nanodrop Technologies, Wilmington, DE, USA) and only samples determined to have A260/280 absorbance ratios between 1.8 and 2.2 were considered for further analyses. The integrity of the total RNA was evaluated by denaturing gel electrophoresis on a 0.9% (w/v) agarose gel with formaldehyde (10 mL of 10× 3-morpholinepropane sulfonic acid [MOPS] running buffer) and 18 mL of 37% formaldehyde (12 mol/L) in pH 7.0 1× MOPS running buffer (0.4 mol/L MOPS, 1 mol/L sodium acetate, and 0.01 mol/L EDTA) after the RNA treatment at 65 °C for 10 min. To remove any contamination of gDNA, 1 µg of the RNA sample was treated with dsDNase (EN0771, Thermo Fisher Scientific) (final volume 40 µL) and, thereafter, RNA was reverse transcribed to cDNA at 42 °C for 60 min with random hexamer primers using the RevertAid RT Kit (EP0441; Thermo Fisher Scientific) according to the manufacturer’s instructions.
The end-point PCR amplification of the putative bsh gene was carried out with a Dream Taq DNA polymerase. RT-PCR of the 16S rRNA gene was used as the positive control and carried out as previously reported [57 (link)]. RT-qPCR reactions were performed with tenfold-diluted cDNA using the PowerUp SYBR Green Master Mix (A25742; Thermo Fisher Scientific) on a QuantStudio 3 real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Each reaction was prepared in a 20 μL mixture containing 10 μL of the Power SYBR Green master mix, 0.3 µM of each primer with the designated final concentration, and 5 μL of diluted cDNA. The thermal conditions were as follows: 50 °C for 2 min, 95 °C for 2 min, 40 cycles at 95 °C for 15 s, and then at 60 °C for 1 min with fluorescence measurement, and the melt curve stage including 95 °C for 15 s, 60 °C for 1 min, and increasing the temperature step to 95 °C at a rate of 0.15 °C/s. All the primers used in this study are listed in Supplementary Table S3. The expression of the bsh gene was normalized to that of the 16S rRNA gene to yield a relative transcript level. Gene expression ratios were calculated using the software tool REST 2009 based on the efficiency-corrected method [58 (link)]. All qPCR reactions were performed in triplicates including the non-template control for each target.

In this study, we sequenced and assembled the complete plastid genome of I. orchioides. Young I. orchioides leaves were collected from Yangiabad, Uzbekistan (N 41°08′32.2″, E 70°07′30.4″). The voucher specimen was prepared and deposited at the Herbarium of the Korea National Arboretum (KH) under accession number KHB1544459. We identified the species according to the key morphological characters described by Sennikov et al. [38 ]. The collected leaf samples were quickly dried with silica gel in a zip-lock plastic bag and stored at room temperature until further use. Genomic DNA was isolated using the DNeasy Plant Mini Kit, following the manufacturer’s protocol (Qiagen, Hilden, Germany). We checked the quantity of the isolated DNA using a NanoDrop ND1000 system (Thermo Fisher Scientific, MA, USA; quality cutoff, OD 260/280 ratio between 1.7–1.9). The isolated DNA was visualized using 1% agarose gel electrophoresis. We constructed the Illumina paired-end libraries of I. orchioides with insert sizes of 300 bp and sent to LABGENOMICS (http://www.labgenomics.co.kr, Sungnamsi, Korea) for sequencing. The prepared libraries were sequenced on MiSeq platform (Illumina Inc., San Diego, CA, USA). We filtered poor quality reads (Phred score, Q < 20) with trim function implemented in CLC Assembly Cell package v. 4.2.1 (CLC Inc., Denmark).

Total RNA from 6 samples (one skin piece per sheep for each color) was separated using TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA) and genomic DNA was eliminated by DNase I (Roche Diagnostics, Chicago, IL, USA) according to the manufacturer’s instructions. The purity and concentration of RNA were evaluated by the NanoDrop ND-1000 system (Thermo Fisher Scientific, Wilmington, DE, USA). The integrity of RNA was confirmed using agarose gel electrophoresis. The purified RNA (25 μg) was fragmented at 70°C for 6 min using RNA Fragmentation Buffer (100 mM ZnCl₂, 100 mM TrisHCl). Most fragmented RNA (98%) was incubated with m⁶A-specific antibody (Synaptic Systems, Gottingen, Germany), and collected for immunoprecipitation (IP) and the rest (2%) for IP control (Input). The m⁶A-enriched RNA and input RNA (2 μL) were reversed to cDNA, second-strand DNA was synthesized, added with dUTP and A-base, and finally, a library was formed by polymerase chain reaction (PCR) amplification. Then the obtained libraries were sequenced on an Illumina Novaseq 6000 platform.

Sample preparation and DNA (deoxyribonucleic acid) extraction.
The swabs were incubated for 18 to 24 h into Todd Hewitt selective medium containing gentamicin and nalidixic acid. After centrifugation of the broth, the precipitate was washed with 1X PBS (phosphate-buffered saline) solution (pH = 7.2) and centrifuged again. Then, the precipitate was washed with 1X Tris-EDTA (TE) buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH = 7.5), and DNA extracted by thermal lysis. The thermal lysis protocol was performed using TE solution for 15 min at 100 °C followed by 15 min at − 80 °C to lyse bacterial cell walls. The quality and quantity of DNA extracted from samples were estimated spectrophotometrically in a Nanodrop ND-1000 system (Thermo Fisher Scientific, USA), at 260 nm (A260) and 280 nm (A280) absorbance, with the sample diluted to 5 ng/μL.