Nanodrop nd spectrophotometer

RNA was isolated from RNAp preserved blood samples using the Qiagen RNAeasy Plus kit that includes a gDNA eliminator column (Qiagen, Germany) per the manufacturer’s instructions. After determination of the RNA concentration using a NanoDrop ND spectrophotometer (Thermo Fisher Scientific Inc, USA), the isolated nucleic acid was treated to remove residual DNA with a DNA-free kit (Ambion, Life Technologies, USA). The treated RNA was then transcribed to cDNA with the QuantiTect Reverse Transcription Kit (Qiagen, Germany) following the manufacturer’s instructions.

Clinical samples were collected in phosphate buffer solution and were kept in this solution less than 3 min, followed by immediate immersion in the tissue stabilization solution RNAlater (Ambion, Applied Biosystems, Foster City, CA, USA). The solution containing the tissue was kept overnight at 4°C and subsequently stored at −80°C. Tissue was homogenized in 1 mL of TRI reagent (Molecular Research Center, INC., Cincinnati, OH, USA). RNA extraction was performed following the instructions of the supplier until precipitation with 95% ethanol (Sigma-Aldrich, St. Louis, MO, USA). The solution was then placed in the column provided in the SV Total RNA isolation kit (Promega, Madison, WI, USA) and the instructions of the supplier were followed. Genomic DNA was digested using the DNase I enzyme supplied in the kit. Quantity and purity of the RNA were determined by measuring the optical density of each sample at 260 and 280 nm using a NanoDrop ND Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).

Genomic DNA was extracted from algal culture samples with associated microbiota using a ZR Fungal/Bacterial DNA MiniPrep (ZYMO Research, Irvine, CA) following the manufacturer's protocol. 16S gene PCR preparation with standard procedures with barcoded primer set 341F forward and 518R reverse primer as previously described (Bartram et al., 2011 (link)). The triplicate PCR products from each sample were pooled and purified using QIAquick PCR purification kit (Qiagen, Valencia, CA) followed by quantification on a Nanodrop ND spectrophotometer (Thermo Science, Wilmington, DE). Twenty-three samples in equal amount with unique index sequence were mixed and further subjected to 2% gel purification using QIAquick gel extraction kit (Qiagen) following by quantification with a Bioanalyser DNA 7500 chip (Agilent Technologies, Santa Clara, CA). The prepared 16S rRNA gene library with addition of 30% PhiX was sequenced for 151-nucleotide paired-end multiplex sequence on MiSeq (Illumina, Hayward, CA) with a loading concentration of 8 pM following manufacturer's protocol.