Nanodrop onec spectrophotometer

Cell media and cell extract supernatants containing RBDmfc proteins were purified using a MabSelect protein A resin (GE Healthcare, Uppsala, Sweden; 17819902) on an AKTA FPLC Basic 10 (GE Healthcare, Uppsala, Sweden). Resin was equilibrated in PBS pH 7.2, supernatants or cellular extracts were loaded onto the resin and washed with 20 column volumes (CV) PBS pH 7.2. RBDmfc proteins bound to the resin were eluted with 10 CV of a proprietary acidic buffer. Eluted fractions were concentrated on Vivaspin 20 (10,000 molecular weight [MW] cutoff filtration units; Sartorius, Göttingen, Germany; VS2002) and dialysed against PBS pH 7.2 using the same Vivaspin 20 filtration unit.
Concentration of the purified RBDmfc proteins was measured with a NanoDrop One C spectrophotometer (ThermoFisher Scientific, Carlsbad, CA, USA) at 280 nm. Protein concentrations were calculated using a UV extinction coefficient ε (0.1%, 280 nm) of 1.357 for WT RBDmfc protein and 1.386 for B.1.351 RBDmfc protein. UV extinction coefficients were determined by computation using the ExPASy ProtParam tool (https://web.expasy.org/protparam, accessed on 23 August 2022), according to the amino acid composition of the respective proteins and assuming that all pairs of cysteine residues form cystines (i.e., are joined by a disulphide bond).

Total RNAs were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) as previously described in our recent study [32 (link)]. A NanoDrop One^C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) was used to ascertain RNA concentration. First-strand cDNA was synthesized from 1.0 μg of total RNA in 20 μL volume using the PrimeScript reverse transcriptase (RT) kit (containing gDNA Eraser, Perfect Real Time, TaKaRa, Dalian, China) according the manufacturer’s recommendations. The cDNA was diluted 10-fold for the subsequent general polymerase chain reaction (PCR), reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR), and dsRNA synthesis experiments.

qRT-PCR was used to study the effect of Gin on the transcription of biofilm-regulated genes of E. coli. E. coli was incubated with and without Gin for 24 h at 37 °C. Bacterial RNA Kit (Omega, Norcross, GA, USA) was used to extract total RNA. RNA quantity was measured by NanoDrop One^C spectrophotometer (Thermo Scientific, USA) and RNA quality was determined via agarose gel electrophoresis. Further, the RNA was reverse-transcribed into cDNA with a PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA Corporation, Kusatsu, Japan). The TB Green^® Premix Ex TaqTM II (Tli RNaseH Plus) (TAKARA Corporation, Kusatsu, Japan) was used for qRT-PCR. The 2−∆∆Ct method was used to assess relative changes in gene transcription levels. The gapA gene was used as an internal control [83 (link)]. In Table S2, the primers used in the current study are listed.

Total RAN was isolated using the RNase Mini Kit (Qiagen). RNA concentrations were determined using a Nanodrop One^c spectrophotometer (Thermo Fisher Scientific). In total, 100 ng of total RNA was used for reverse transcription to make cDNA using iScript Reverse Transcription kit (Bio-Rad, 1708841). Gene expression was assessed by SYBR green on QuantStudio 7 (Applied Biosystems). Data were normalized to GAPDH or β-actin using the ΔΔCt method. As a standard practice for qPCR experiments, no template and no RT controls were always included for quality control purposes. Detailed primer information is provided in Supplemental Table 1.

Fecal samples from the participants were collected using a fecal collection kit and delivered on ice bags before and after intervention. Samples were frozen and stored at −20 °C until use. DNA was extracted using a QIAamp Fast DNA Stool Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. The purity of the DNA samples was quantified by measuring the A260/280 ratio with the Nanodrop OneC spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). DNA samples were frozen and stored at −20 °C until use. 110 DNA samples were sent to Novogene Bioinformatics Technology Co., Ltd. (Beijing, China) for paired-end 16S rRNA gene sequencing. The quality and quantity of samples were checked using an Agilent 5400 Bioanalyzer (Agilent, Santa Clara, CA, USA) before library preparation. The V4 region of the 16S rRNA gene was amplified with PCR using 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers connected with barcodes [33 (link)]. 16S rRNA gene sequencing (2 × 250 bp PE V4) was performed on an Illumina Novaseq 6000 platform.

Microbial genomic DNA was extracted from both shells and internal organs of lobsters using the DNeasy Blood & Tissue kit (Qiagen) according to the manufacturer’s protocols, with a minor adjustment in the pretreatment of the shells. Briefly, shells in RNAlater stabilization solution were cut into small square pieces, placed in a microcentrifuge tube in 320 μl of PBS, and incubated on ice for 1 h to help detach bacteria from the shells. The incubated shells in PBS were disrupted and homogenized using a handheld homogenizer (Bel-Art). The resulting homogenizing solution, which contains bacteria from the surface of shells as well as bacteria present in the shells, was subsequently used for DNA extraction. For internal organs, 10–25 mg of the hepatopancreas, intestine, green gland, and testis were used for the extraction of DNA according to the manufacturer’s instructions. The DNA concentration and purity from all samples were measured by a NanoDrop One^C spectrophotometer (Thermo Scientific).