Sample collection
Sediment cores were collected during IODP Expedition 385 using the drilling vessel JOIDES Resolution. Holes at each site were first advanced using advanced piston coring (APC), then half-length APC, and then extended core barrel (XCB) coring as necessary. Temperature measurements used the advanced piston corer temperature (APCT-3) and Sediment Temperature 2 (SET2) tools8. Downhole logging conducted after coring used the triple combination and Formation MicroScanner sonic logging tool strings. After bringing core sections onto the core receiving platform of the D/V JOIDES Resolution, whole round samples for microbiology were retrieved within ~30 minutes using ethanol-cleaned spatulas. Samples for biogeochemical measurements were obtained and processed shipboard (Teske et al., 2021). Whole round samples for DNA-based studies were capped with ethanol-sterilized endcaps, transferred to the microbiology laboratory, and stored briefly at 4 °C in heat-sealed tri-foil gas-tight laminated bags flushed with nitrogen until processing. Masks, gloves and laboratory coats were worn during sample handling in the laboratory where core samples were transferred from their gas-tight bags onto sterilized foil on the bench surface inside a Table KOACH T 500-F system, which creates an ISO Class I clean air environment (Koken Ltd., Japan). In addition, the bench surface was targeted with a fanless ionizer (Winstat BF2MA, Shishido Electrostatic Co., Ltd., Japan). Within this clean space, the exterior 2 cm of the extruded core section were removed using a sterilized ceramic knife. The core interior was transferred to sterile 50-mL Falcon tubes, labeled, and immediately frozen at –80 °C for post cruise analyses. For RNA-based studies, sampling occurred immediately after core retrieval on the core receiving platform by sub-coring with a sterile, cutoff 50cc syringe into the center of each freshly cut core section targeted. These sub-cores were immediately frozen in liquid nitrogen and stored at –80 °C.

DNA extraction and sequencing
DNA was extracted from selected core samples using a FastDNA SPIN Kit for Soil (MP Biomedicals). Up to 5 grams of sediment were processed following a modified manufacturer’s protocol (Ramirezet al., 2018). Briefly, each sediment sample was homogenized twice (vs. once that the manufacturer suggests) in Lysing Matrix E tubes for 40 seconds at speed 5.5 m/s, using the MP biomedicals bench top homogenizer equipped with 2 ml tube adaptors. Between the two homogenization rounds the samples were placed on ice for 2 minutes. After the second homogenization the samples were centrifuged at 14,000 x g for 5 minutes. For each sample, the supernatant and the top layer of the pellet was transferred to a clean 2 ml tube where proteins were precipitated by the addition of the protein precipitation solution (PPS) provided in the extraction kit. The rest of the extraction protocol followed the manufacturer’s recommendations. When parallel extractions were performed, the extracts were pooled and concentrated using EMD 3kDa Amicon Ultra-0.5 ml Centrifugal Filters (Millipore Sigma). A control extraction, in which no sediment was added, was included to account for any laboratory contaminants. All libraries for metagenome sequencing (n = 29; 26 samples and 3 controls) were prepared from genomic DNA extracts that were submitted at the University of Delaware DNA Sequencing & Genotyping Center. Thirteen libraries were sequenced with NovaSeq S4 PE150 (Illumina) at the University of California, Davis Genome Center, and thirteen libraries were sequenced with NextSeq550 (Illumina) at the University of Delaware DNA Sequencing & Genotyping Center. Metagenome sequence reads were deposited to the National Center for Biotechnology Information Sequence Read Archive under access numbers SRR23614663-23614677 and SRR22580794-SRR22580807 (Bioproject PRJNA909197).

Prokaryotic (bacterial and archaeal) 16S rRNA gene amplification and Illumina MiSeq sequencing
The 16S rRNA gene V4/V5 hypervariable regions were targeted using the general prokaryotic primer pair 515F-Y (5′-GTGYCAGCMGCCGCGGTAA-3′; Parada et al., 2016) and 926 R (5′-CCGYCAATTYMTTTRAGTTT-3′; Quince et al., 2011) to recover 16S rRNA gene fragments of both Bacteria and Archaea. Libraries for samples 1547B-1H2, 1547B-3H2, 1545B-1H2, and 1545B-6H2 were prepared from DNA extracts by the Georgia Genomics and Bioinformatics Core (GGBC) at the University of Georgia. Libraries for all other samples and control fluid filters were prepared internally through the amplification steps described below, before sent to GGBC (for Illumina MiSeq) or the University of Delaware DNA Sequencing & Genotyping Center for final library preparation and sequencing. Illumina MiSeq overhang adapter sequences were added to locus-specific primers for use in first round Polymerase Chain Reaction (PCR) amplifications. 16S rRNA PCR amplifications were performed for each sample (1:10 dilution) in triplicate using SpeedStar™ HS DNA Polymerase (TaKaRa) and 10X Fast Buffer I as described by the manufacturer. Thermocycling conditions were: 95 °C for 5 min; x30 (95 °C for 30 s, 60 °C for 30 s, 72 °C for 60 s); 72 °C for 5 min and a 4 °C hold. PCR amplification replicates were combined and purified with AMPure® XP beads (Beckman Coulter). Extraction kit control amplifications were attempted at both Laboratories where extractions were physically performed with only one yielding an amplicon sufficient for library construction. All libraries produced with 515F-Y/926 R amplified fragments were sequenced on an Illumina MiSeq platform with PE300 read lengths.

Archaeal 16S rRNA gene amplification and PacBio sequencing
To better capture the diversity of the subsurface archaeal community, we used the archaeal 16S rRNA primer set Arch25F and Arch806R that targets the V2-V4 hypervariable regions and generate a ~800 base pairs 16S rRNA amplicon. The use of larger 16S rRNA gene amplicons provides an improved basis for phylogenetic identifications, and alternate primers reduce the dependency on the extremely widespread Prokaryotic Miseq primer set (Parada et al., 2016) both factors are essential for the detection of rare or novel phylogenetic lineages41. The primer set is Arch25F (5′TCYGKTTGATCCYGSCRG 3′; Urbach et al., 2001) and Arch806R (5′GGACTACVSGGGTATCTAAT 3′; Takai et al., 2000). The 806R primer site is unusually conserved among archaea, including uncultured subsurface lineages; Teske et al., 2018). PCR reactions were performed using the SpeedSTARTM HS DNA Polymerase (TaKaRa) kit with the following modifications: each 25 μΜ PCR reaction contained up to 1 ng of template DNA, 2X Fast Buffer I, 2.5 mM dNTP mixture, 5 units of SpeedSTAR HS DNA Polymerase, 10 mM of each primer and DEPC water (Fisher BioReagents™) up to 25 μΜ. The PCR reactions were performed in an Eppendorf Mastercycler Pro S Vapoprotect (Model 6321) thermocycler with the following conditions: 95 °C for 5 min, followed by 30 cycles of 94 °C (30 s), 55 °C (30 s), 72 °C (45 s). The total volume of PCR reactions was run in 2% agarose gel (Low-EEO/Multi-Purpose/Molecular Biology Grade Fisher BioReagents™) and the correct size PCR products (~800 bp), were isolated and recovered from the gel using the Zymoclean Gel DNA Recovery Kit as instructed by the manufacturer. Libraries for PacBio sequencing were prepared from the recovered and gel purified DNA extracts at the University of Delaware DNA Sequencing & Genotyping Center.

RNA extraction and sequencing
Total RNA was extracted successfully from 19 sediment samples from sites U1545B-U1552B. Before each RNA extraction, all samples including a blank sample (control), were washed twice with absolute ethanol (200 proof; purity ≥ 99.5%; Thermo Scientific Chemicals), and one time with DEPC water (Fisher BioReagents) to remove hydrocarbons and other inhibitory elements present in Guyamas sediments, that without these washes, resulted in low or zero RNA yield. In brief, 13–15g of frozen sediments were transferred into UV-sterilized 50 ml Falcon tubes (RNAase/DNase free) using clean, autoclaved and ethanol-washed metallic spatulas. Each tube received an equal volume of absolute ethanol and was shaken manually for 2 min followed by 30 s of vortexing at full speed to create a slurry. Samples were transferred into an Eppendorf centrifuge (5810 R) and were centrifuged at room temperature for 2 min at 2000 rpm. The supernatant was decanted, and the ethanol wash was repeated. After decanting the supernatant of the second ethanol wash, an equal volume of DEPC water was added into each sample. Samples were manually shaken and vortexed as before to create slurry, and were transferred into the Eppendorf centrifuge (5810 R) where they were centrifuged at room temperature for 2 min at 2000 rpm. The supernatant was decanted, and each sediment sample was immediately divided into three bead-containing 15 mL Falcon tubes, provided by the PowerSoil Total RNA Isolation Kit (Qiagen). RNA was extracted as suggested by the manufacturer with the modification that the RNA extracted from the three aliquots was pooled into one RNA collection column and eluted at 30 μl final volume. All RNA extractions were performed in a UV-sterilized clean hood (two UV cycles of 15 min each) that was installed with HEPA filters. Surfaces inside the hood and pipettes were thoroughly cleaned with RNase AWAY (Thermo Scientific) before every RNA extraction and in between extraction steps. Trace DNA contaminants were removed from RNA extracts using TURBO Dnase (Thermo Fisher Scientific) and the manufacturer’s protocol. Removal of DNA from the RNA extracts was confirmed with PCR reactions using the bacterial primers BACT1369F/PROK1541R (F: 5ʹCGGTGAATACGTTCYCGG 3ʹ, R: 5ʹAAGGAGGTGATCCRGCCGCA 3ʹ; Parada et al., 2016), targeting the small ribosomal subunit (SSU) of 16S rRNA gene. Each 25 μl PCR reaction was prepared using GoTaq G2 Flexi DNA Polymerase (Promega) and contained 0.5 U μl−1 GoTaq G2 Flexi DNA Polymerase, 1X Colorless GoTaq Flexi Buffer, 2.5 mM MgCl2, (Promega) 0.4 mM dNTP Mix (Promega), 4 μM of each primer (final concentrations), and DEPC water. These PCR amplifications were performed in an Eppendorf Mastercycler Pro S Vapoprotect (Model 6321) thermocycler with following conditions: 94 °C for 5 min, followed by 35 cycles of 94 °C (30 s), 55 °C (30 s), and 72 °C (45 s). The PCR reaction products were run in 2% agarose gels (Low-EEO/Multi-Purpose/Molecular Biology Grade Fisher BioReagents) to confirm absence of DNA products. RNA quantification (ng μl−1) was performed using Qubit RNA High Sensitivity (HS), Broad Range (BR), and Extended Range (XR) Assay Kits, (Invitrogen).

Amplified cDNAs from the DNA-free RNA extracts were prepared using the Ovation RNA-Seq System V2 (Tecan) following manufacturer’s suggestions. cDNAs were submitted to the Georgia Genomics and Bioinformatics Core for library preparation and sequencing using NextSeq 500 PE 150 High Output (Illumina). The sequencing of the cDNA library from the control sample was unsuccessful as it failed to generate any sequences that met the length criterion of 300-400 base pairs.