Nanodrop nd 1000 spectrophotometer

Total RNA was extracted from 100 mg of ground leaf tissue using previously reported protocols^{63 (link),64} . RNA was purified using RNeasy® Minelute Cleanup Kit (QIAGEN). Removal of any residual genomic DNA in RNA samples was verified by PCR amplification of total RNA (with no cDNA synthesis step) using the designed primers listed in Table 1. RNA samples containing DNA were further DNase treated until no PCR amplification of RNA samples was obtained. Prior to retrotranscription the concentration and integrity of RNA were verified by an optical density at 260 nm (OD260)/OD280 absorption ratio in a NanoDrop ND- 1000 spectrophotometer (Thermo scientific).
First and second-strand of complementary DNA (cDNA) were synthesized using SuperScript® III First-Strand (Invitrogen) and DNA Polymerase I (BioLabs), respectively. cDNA was cleaning by QUIquick PCR Purification Kit (QIAGEN and DNase treated by the RNase-Free DNase Set (Qiagen), according to the manufacturer’s recommendations. Conventional RT-PCR and PCR assays followed by gel electrophoresis were performed to verify the amplification of cDNA using the designed primers. Quality and quantity of cDNA was determined by running aliquots in agarose gels and by spectrophotometric analysis in a NanoDrop ND-1000 spectrophotometer (Thermo scientific).

Total RNA was isolated using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions and treated with DNase I, RNase-free (Thermo Fisher, USA). The RNA concentration was quantified using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher). The RNA integrity was determined by agarose formaldehyde gel electrophoresis and using the Agilent Bioanalyzer platform 2100 (Agilent, USA). Purity and concentration were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher). A total of 12 libraries (Δmus-52 and Δpac-3 strains, cultivated in high- and low-Pi concentration media, each, in three biological replicates) were sequenced on an Illumina HiSeq2000 (Illumina, USA) sequencer platform with paired-end 100-bp reads. RNA-seq data were analyzed and validated as previously described (Martins et al., 2019 (link)) and deposited at the GEO database with accession number GSE132373.

For RNA extraction, PBMC samples stored in RNAlater (approximately 1 × 10⁶ cells) were centrifuged at 2300 × g for 15 min. The supernatant of RNAlater was removed, and remaining cells were homogenised and extracted using the RNeasy Mini Kit (Qiagen, Doncaster, VIC, Australia) following manufacturer’s instructions. To remove contaminating DNA, RNA preparations were treated with DNase I following manufacturer’s instructions (Applied Biosystems, Carslbad, CA, USA). The concentration and purity of RNA was assessed using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). RNA was immediately processed for cDNA synthesis or stored at −80°C.
Reverse transcription was performed using the Revertaid First Strand cDNA Synthesis kit (Thermo Scientific, Lithuania) according to the manufacturer’s instructions, using oligo dT primers. Due to biological variation, 70–700 ng of RNA was used (in 10 μl) in each reaction. To control for contamination for genomic DNA, ‘no reverse transcriptase’ (NRT) controls were made using the same protocol omitting reverse transcriptase. cDNA concentration was determined using a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and then cDNA was stored at −20°C until use in qPCR.

Genomic DNA was purified from homogenized oyster powders using the Macherey-Nagel genomic DNA-from-tissue kit (catalog number 740952.250) following the manufacturer’s instructions, with an additional step of RNase A treatment (Macherey-Nagel; catalog number 740505). The quality and quantity of genomic DNA were estimated on a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). Total DNA was resuspended to a final concentration of 20 ng μl⁻¹ prior to qPCR. Total RNA was extracted from oyster pool powders (10 mg) homogenized in 500 μl of Tri-Reagent (Invitrogen) by vortexing for 2 h at 4°C. Prior to extraction, insoluble materials were removed by centrifugation at 12,000 × g for 10 min at 4°C, and supernatant was incubated with 100 μl of chloroform at room temperature for 3 min. After centrifugation at 12,000 × g for 15 min at 4°C, total RNA in the aqueous phase was extracted using the Direct-Zol RNA miniprep kit (Zymo Research; reference no. R2052) according to the manufacturer’s protocol. Quantity and purity of total RNAs were checked using the NanoDrop ND-1000 spectrophotometer (Thermo Scientific) and capillary electrophoresis (Agilent BioAnalyzer 2100).

Isolation of total RNA from cell lines was conducted using an RNeasy® Micro Kit (QIAGEN, Tokyo, Japan). The RNA concentration was quantified using a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific). Then, synthesis of cDNA from total RNA was performed using an RNA-to-cDNA kit (Applied Biosystems). The quality and quantity of the cDNA samples were evaluated via standard electrophoresis and a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific).

Five of seven replications were randomly selected, and fifty caecal content samples from 21-day-old broilers and fifty caecal content samples from 42-day-old broilers were used for DNA extraction using the QIAmpH DNA Stool Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. A Nanodrop ND-1000 spectrophotometer (Thermo Scientific, USA) was used to assess the DNA quantity of the DNA samples. According to the DNA concentration, equal quantities of two DNA samples extracted from two caecal content samples from the same replicate were pooled into one DNA sample for further sequencing. The quantity and quality of the 40 pooled DNA samples (20 samples from 21-day-old broilers and 20 samples from 42-day-old broilers) was further assessed by a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, USA), and then, the samples were stored at − 80 °C until sequence analysis.