The methods characterized below are part of the following publication, currently in prep: Tucker SJ, Fuessel J, Freel KC, Kiefl E, Freel EB, Ramfelt O, Sullivan CE, Gajigan AP, Mochimaru H, Souza MR, Quinn M, Ratum C, Tran LL, Miller SE, Trigodet F, Lolans K, Morrison HG, Fallon B, Huettel B, Pan T, Rappé MS, Eren AM. A high-resolution diel survey of surface ocean metagenomes, metatranscriptomes, and transfer RNA (tRNA) transcripts.

Hawaiʻi Diel Sampling (HaDS) collection and processing:

We sampled surface seawater from August 18, 2021 9:00 AM to August 20, 2021 9:00 AM Hawaiʻi Standard Time (HST), from a depth of ~0.25 m at station HP1 within coastal Kāneʻohe Bay, Oʻahu, Hawaiʻi, and station STO1 in the adjacent offshore. Sampling took place every 1.5 hours for 48 hours and yielded 33 sampling events at each site. We collected seawater samples for flow cytometry, fluorometric chlorophyll a concentrations, and nucleic acids, and recorded in situ measurements of seawater temperature, pH, and salinity with a YSI 6600 or YSI EcoSense EC300 (YSI Incorporated, Yellow Springs, OH, USA) and and in situ measurements of Photosynthetic Photon Flux Fluence Rate (PPFFR) with a LI-193 Underwater Spherical Quantum Sensor and LI-250A Light Meter (LI-COR Environmental, Lincoln, NE, USA).

Roughly 10-20L of seawater was first pre-filtered through an 85-μm Nitex mesh and then ~4 (HP1) or ~9 (STO1) L of seawater was filtered through 0.2-μm pore-sized polyethersulfone (PES) Sterivex cartridge filters (MilliporeSigma, Burlington, MA, USA) to collect microbial cells for nucleic acids isolation. RNAlater (Invitrogen, Waltham, MA, United States) was immediately added to the Sterivex filters and stored the samples at −80 °C until further processing.

Seawater subsamples (125 mL) were collected on 25-mm diameter GF/F glass microfiber filters (Whatman, GE Healthcare Life Sciences, Chicago, IL, USA) and the filters were stored in aluminum foil at −80 °C until analysis of fluorometric chlorophyll a concentrations. Chlorophyll a was extracted in 100% acetone and the concentration determined with a Turner 10-AU fluorometer (Turner Designs, Sunnyvale, CA, USA), according to standard techniques (Welschmeyer 1994). Seawater aliquots (2 mL) in a final concentration of 0.95% (v:v) paraformaldehyde (Electron Microscopy Services, Hatfield, PA, USA) were collected and stored at −80 °C for cellular enumeration. Abundances of heterotrophic bacteria, photosynthetic picoeukaryotes, Synechococcus, and Prochlorococcus were enumerated by the SOEST Flow Cytometry Facility on a Beckman Coulter CytoFLEX S, following the method of Monger and Landry (1993). Dissolved inorganic nutrients were measured by the SOEST Laboratory for Analytical Biogeochemistry (S-LAB) on a Seal Analytical AA3 HR Nutrient Autoanalyzer, while concentrations of dissolved organic carbon and total nitrogen were measured by the S-LAB on a Shimadzu High-Temperature TOC-L Combustion Analyzer.

DNA extraction, preparation, and sequencing for short-read metagenomics:

Co-extraction of RNA and DNA was adapted from previously published protocols (Donaldson et al. 2020) and was used to extract DNA from Sterivex cartridge filters for short-read metagenomic sequencing. Cells suspended in the RNAlater within the Sterivex filter cartridges were collected by attaching a 10 mL air-filled clean syringe to the Sterivex cartridge and pushing the liquid into a nuclease-free 1 mL centrifuge vial. After centrifugation for 15 min at 4 °C at 13,000 rpm, we discarded the supernatant and kept the cell pellet on ice for further processing. The cartridge was opened holding the filter with a sterilized PVC pipe cutter and the cylinder with the PES membrane removed. Using a sterilized scalpel, the filter membrane was removed from the barrel. We cut the filter into ~6-8 small pieces in a sterile petri dish and transferred the pieces to a Lysing Matrix E (MP Biomedicals, Irvine, CA, USA) vial and added 450 µL Buffer A, 210 µL 20% SDS and 500 µL phenol:chloroform:IAA 125:24:1 pH 8. (50 mL Buffer A: 2 mL 5 M NaCl, 2 mL 0.5 M EDTA, 46 mL dH2O, all solutions were molecular grade and nuclease-free). Using in 50 µl Buffer A, we resuspended the cell pellet obtained from RNAlater and vortexed vigorously for 30 sec followed by a quick centrifugation step in a microcentrifuge and transferred the solution to the Lysing Matrix E vial. The cells were lysed with a Bead Ruptor Elite (OMNI International, Kennesaw, GA, USA) on setting 6 for 30 sec followed by two min on ice followed by another lysing cycle with the same settings. The vials were centrifuged for 3 min at 4 °C at 13,000 rpm and the upper aqueous phase removed to a fresh, nuclease-free 1.5 mL centrifuge vial. Following the addition of 500 µL phenol:chloroform:IAA, we repeated the centrifugation step. After transferring the aqueous phase to a new centrifuge vial, we added 3 M sodium acetate at 1/10th of the transferred volume and mixed the solution by inversion. Following the addition of 500 µL ice-cold pure ethanol, we placed the samples in the freezer at -20 °C overnight.

The next day, samples were thawed and centrifuged at 13,000 rpm at 4 °C for 20 min, and the supernatant was carefully decanted to retain the nucleic acid pellet. We washed the pellet with 500 µL 70% ethanol at 4 °C and vortexed shortly followed by centrifugation at 13,000 rpm at 4 °C for 10 min. We decanted the liquid carefully and air-dried the pellet for 15 min. We added 100 µL of nuclease-free dH2O to the pellet and vortexed the pellet for 20 min with a Vortex-Genie 2 (Scientific Industries, Bohemia, NY, USA) and a Vortex adapter 24 (Qiagen, Hilden, Germany). We purified the DNA with the DNA Clean & Concentrator-100 (Zymo Research, Irvine, CA, USA) and eluted in a final volume of 30 µL.

Following shearing of DNA to 400 bp on a Covaris S220 focused-ultrasonicator system, DNA was cleaned and concentrated with AMPure XP beads (Beckman Coulter, Brea, CA, USA) and metagenomic sequencing libraries prepared using the Ovation Ultralow System V2 with indexed sequencing adapters (Tecan Genomics, Männedorf, Switzerland). Final libraries were pooled at equimolar amounts and size-selected the library using a BluePippin 1.5% agarose gel cassette (Sage Science, Beverly, MA, USA) targeting the 468–572 bp region. DNA concentration was evaluated using the Qubit dsDNA high-sensitivity (HS) assays (Thermo Fisher Scientific) and the purity estimated with a NanoDrop Microvolume Spectrophotometer (Thermo Fisher Scientific) before library preparation. We quantified the short-read metagenomic libraries using real-time PCR using KAPA library quantification reagents (Roche 07960204001) prior to sequencing. We paired-end sequenced the final pool on a NextSeq 500/550 High Output v2.5 using the 300 cycles kit (Illumina, San Diego, CA, USA). The Keck Sequencing Facility at the Marine Biological Laboratory in Woods Hole, MA, USA carried out the short-read metagenomic library preparation and sequencing.

Nucleic acid extraction, preparation, and sequencing for long-read metagenomics and metatranscriptomics:

The filters were first removed from the Sterivex cartridge as described above, and cut into small pieces to transfer them to 2 mL ZR BashingBead lysis tubes with 0.1 & 0.5 mm beads (Zymo Research Europe, Germany) for subsequent cell disruption with a Qiagen TissueLyser (3 min. at 25 Hz). Next, DNA and total RNA were isolated from the same starting material using the ZymoBIOMICS DNA/RNA Miniprep Kit (Zymo Research Europe, Germany), with an additional DNase I treatment of the extracted RNA.

DNA directly served as input for a barcoded DNA library preparation without prior fragmentation. DNA was quality-controlled using HS Qubit assays (Thermo, Waltham, USA) and pulse-field capillary electrophoresis (Agilent FEMTOpulse). We prepared HiFi SMRTbell libraries (PacBio, Menlo Park, CA, USA) for long-read DNA sequencing following manufacturer protocols using the Ultra Low DNA Input approach and sequenced the libraries on a PacBio Revio device. For demultiplexing, adapter trimming, and deduplication of the sequencing data we relied on the PacBio SMRTlink software suite.

RNAseq libraries were prepared with the Universal Prokaryotic RNA-Seq Prokaryotic Any Deplete Kit by Tecan Genomics, including the proprietary adapter addition step of the kit and dual 8-mer barcode indexing. RNA was evaluated quality using HS Qubit assays (Thermo, Waltham, USA) and a PicoChip (Agilent Bioanalyzer) for total RNA. The libraries were paired-end sequenced on an Illumina NextSeq2000. The MPI Cologne Genomics Center conducted nucleic acid extraction, library preparation, and sequencing for long-read metagenomics and metatranscriptomics.

RNA extraction, preparation, and transfer RNA sequencing:

We prepared the following solutions: 1) marine microbe lysis solution: 40 mM EDTA, 50 mM TRIS and 0,75 M sucrose final concentration, 2) lysozyme stock solution: 100 mg/mL in TE buffer pH 8, RNAse free, and 3) Lysis solution 670 µL per sample: 600 µL marine microbe lysis solution, 67 µL lysozyme stock solution, 0.2 µL SUPERase (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA, 20 U/μL). For each Sterivex cartridge, we collected and pelleted cells that were suspended in RNAlater following the same steps described above and then resuspended the pellet with 70 µL of lysis solution. We removed the filter from the Sterivex cartridge using the steps described above, cut the filter into small pieces using a clean scalpel, and transferred the filter pieces to a 14 mL culture tube containing 600 µL of lysis solution. We then added the lysis solution with the resuspended cell pellet to the corresponding 14 mL tube with the filter pieces, and vortexed vigorously for 2 min. The samples were incubated in a 37 °C water bath for 45-60 min with gentle agitation of the samples every 10 min. Next, we added 7.4 µL of proteinase K (800 U/mL, New England Biolabs, Ipswich, MA, USA) and 74 µL of 10% SDS for a final concentration of 1%. The sample was placed in a 37 °C water bath for 2 h with gentle agitation every 10 min. Samples were stored at -80 °C until extraction.

The samples were thawed on ice. Next, we transferred the lysate to Lysing Matrix C tubes (MP Biomedicals, Santa Ana, CA, USA), which contain 1 mm silica lysing beads. We added 400 µL of 0.3 M NaOAc/HOAc, 10 mM EDTA, pH4.8 and an equal volume of acetate-saturated phenol/chloroform and vortexed briefly. The samples were placed in a reciprocating bead beater for 2-min at maximum intensity, pausing between 1 min intervals followed by centrifugation at 13,000 x g for 15 min at 4 °C. The aqueous phase was transferred to a fresh Lysing Matrix C tube and the phenol/chloroform extraction steps repeated. To precipitate the RNA, we added 1 v of 100 % isopropanol, vortexed each sample thoroughly, and incubated them overnight at -20 °C. The sample was centrifuged at 4 °C for 15 min at 13,000 x g, the supernatant discarded, and the pellet washed with 1 mL of chilled 75% ethanol. Following centrifugation for 5 min at 13,000 x g at 4 °C and removal of the ethanol, we let the RNA pellet air dry for 10 min at room temperature and then redissolved it in 30 µL of an acid-elution buffer (10 mM NaOAc/1 mM EDTA, pH 4.8). The sample was cleaned with the Zymo Research Oligo Clean & Concentrate, and eluted in RNAse-free water. Finally, we added an equivalent amount of acid-elution buffer to each extract to reach a final concentration of 5 mM NaOac/0.05 mM EDTA, pH 4.8. We evaluated the quality and quantity of the RNA used in the tRNA libraries using a Qubit RNA HS assay (ThermoFisher Scientific, USA) and a Nanodrop Microvolume Spectrophotometer (ThermoFisher Scientific, USA). Previously published protocols for transfer RNA library preparation (MSR-seq; Watkins et al., 2022) were followed with paired-end multiplexed small RNA sequencing conducted on an Illumina NovaSeq 6000. Subsequently, we demultiplexed sequence data with custom scripts.

Kāneʻohe Bay Time-series (KByT) collection and processing:

To contextualize the diel data, we deeply sequenced four metagenomes collected at stations HP1 and STO1 on December 23, 2020 and May 24, 2021. These sampling events were part of the routine Kāneʻohe Bay Time-series sampling and processing of nucleic acids and other associated biogeochemical parameters followed previously published protocols (Tucker et al. 2021, 2025). Briefly, approximately one L of seawater collected from ~2m depth at HP1 and STO1 was prefiltered using an 85 μm Nitex mesh and subsequently collected on a 25-mm diameter 0.1-μm pore-sized polyethersulfone (PES) membrane for nucleic acids (Supor-100, Pall Gelman Inc., Ann Arbor, MI, USA). The filters were submerged in DNA lysis buffer (Suzuki et al., 2001) and stored at −80 °C until extraction. Genomic DNA was extracted using a Qiagen Blood and Tissue Kit (Qiagen Inc., Valencia, CA, USA) with modifications (Becker, Brandon & Rappé, 2007). All other processing of subsamples follow the protocols described above. The short-read metagenomic library preparation and sequencing protocols for these 4 deeply sequenced samples are the same as those used for the diel short-read metagenomes.

Deployment description:

Small boat operations between the Hawaiʻi Institute of Marine Biology, coastal station HP1, and offshore station STO1 were conducted every 1.5 hours for 48 hours between 18 August 2021 and 20 August 2021. Small boat operations conducted through routine Kāneʻohe Bay Time-series (KByT) sampling yielded two additional sampling events at station HP1 and station STO1 on 23 December 2020 and 24 May 2021.