Nanodrop nd 8000

TRI reagent (Sigma) was used to isolate total RNA for mRNA analysis, and miRNeasy kit (Qiagen, Valencia, CA, USA) for miRNA analysis from the snap-frozen tissues or cultured cells. After determining RNA concentration and purity by using NanoDrop ND-8000 (Thermo Fisher Scientific, Waltham, MA, USA), the cDNAs were synthesized by using Reverse Transcriptase M-MLV kit (Invitrogen). The expression levels of miRNA and mRNA were analyzed using SYBR Premix Ex Taq (Takara, Shiga, Japan) in Applied Biosystems ViiATM 7 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Data were analyzed with 2^−ΔΔCT method^{28 (link)} for quantification of the relative mRNA expression levels. Expression values of genes and miRNA were first normalized against GAPDH and small nuclear U6 RNA, and then compared to experimental controls. The primers were purchased from Sangon Biotech (Shanghai, China) and the sequences are listed in Supplementary Table 3.

Total RNA of cultured cells or snap‐frozen tissues was isolated with TRI reagent (Sigma). RNA concentration and purity were measured with NanoDrop ND‐8000 (Thermo Fisher Scientific), and Reverse Transcriptase M‐MLV kit (Invitrogen) was used for cDNAs synthesis. SYBR Premix Ex Taq (Takara) was used to determine expression levels of mRNA by ViiATM 7 Real‐Time PCR System (Applied Biosystems) with 2^−ΔΔCT method. The primers were obtained from Sangon Biotech (Shanghai, China) and the sequences are listed in Table S1.

For HTS analyses, aliquots of water were filtered through 25 mm polycarbonate Isopore membranes (Merck) with 0.2 μm pore size. Filtered volumes ranged between around 500 and 1000 mL, depending on the quantity of suspended particles, and until near-clogging of the filters. Filters were stored at −20°C until DNA extraction with MO BIO PowerWater^® DNA Isolation Kit (MO BIO Laboratories, Qiagen, United States). DNA concentrations, measured with a NanoDrop ND-8000 (Thermo Fisher Scientific, Inc., Waltham, MA, United States), ranged between 5 and 46 ng μL^–1. PCR amplification of bacterial sequences was carried out by targeting a ∼ 460-bp fragment of the 16S rRNA gene variable regions V3–V4 using the specific bacterial primer set 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805Rmod (5′-GACTACNVGGGTWTCTAATCC-3′) with overhang Illumina adapters. The final barcoded library was sequenced on an Illumina^® MiSeq (PE300) platform. A detailed description of procedures is reported in Salmaso et al. (2018a) (link). Sequences in FASTQ format were deposited to the European Nucleotide Archive (ENA) with study Accession No. PRJEB33405.

Sea star tissue and swab samples in 1 mL of TRIzol were removed from the -80°C freezer, placed in an ice bath to thaw, incubated at room temperature for 5 min, and homogenized using sterile RNA free pestles. DNA and RNA were isolated using the manufacturer’s protocol and stored at -80°C until use. The Thermo Scientific Nanodrop ND8000 version 2.2.1 system was used to determine DNA concentration.

Raw sewage samples were collected weekly from 24 WWTPs in Cape Town, South Africa, between 19 May-31 July 2021 (16 weeks). For RNA extraction, 100 mL of grab sample was mixed and centrifuged at 2500 xg for 20 min, whereafter 2.5 mL of the resultant pellet was used for total RNA extraction using a previously described protocol of Johnson et al. (2021). Total RNA was subjected to spectrophotometry using the NanoDrop ND-8000 (Thermo Scientific, USA). Only samples with a A260/280 ratio between 1.8–2 and a A260/230 ratio between 1.8–2.1 was used for subsequent After isolation, the consequent RNA (70 µL) was aliquoted and stored at –80 °C until required for molecular analysis.