SIP Incubation Preparation
IODP Expedition 337 operations commenced July 26 and continued through September 30, 2012 on the D/V Chikyu. Utilizing riser drilling, a sedimentary sequence was recovered down to 2466 m below seafloor (mbsf) at Hole C0020A (41 10' 36" N, 142 12' 02" E) in 1180 m water depth off the Shimokita Peninsula. The drilled sequence transitioned from open marine (youngest; late Pliocene, ~5 Ma) to terrestrial (oldest; late Oligocene, ~30 Ma) with depth. Models for maximum temperature reached by Expedition 337 coring report 63.7 degrees C. Shipboard sedimentological, geochemical, and microbiological data and methods are available through IODP publications. Additional coal petrography is available in Gross et al.  A total of 52 incubation amendment conditions were prepared onboard to interrogate a range of potential deep-biosphere metabolic strategies, and then incubated back in the lab at temperatures approximating that measured in situ. In this study, incubations from shale (Core 8L4; 1606 mbsf; 37C incubation temperature), coal (Core 15R3; 1921 mbsf; 45C incubation temperature), and mixed (homogenized mixture from multiple cores 19R1, 19R5, 19R7, 20R3, 23R6, 23R8, 24R3, 25R1, 25R2, and 25R3; 1950-1999 mbsf; 45C incubation temperature) with methanol and methylamine substrate additions were analyzed. Age estimates of these samples are early to middle Miocene. In situ temperatures ranged from 38C to 48C at these sample depths with pressures ~30 MPa. Two coal beds were included in these incubations: a shallower coal-only sample deposited under more marine-influenced conditions (~1921 mbsf, core 15R3) and a deeper coal bed deposited under more limnic conditions that was included in the mixed lithology sample (~2000 mbsf, cores 24R3 and 25R1). Cores used for incubations were prepared by removal of outer drill-fluid-contaminated layers by sterile ceramic knife as soon as possible after core recovery and stored at 4C until incubation preparation, while maintaining an anaerobic atmosphere during the entire process. For preparation of the SIP incubations, the interior portion of the core was manually crushed into cm-sized pieces under sterile, anaerobic conditions and distributed evenly into sterile 50 ml glass vials with butyl rubber stoppers and screw caps (Nichidenrika-Glass Co. Ltd.).  Vials were flushed with argon and pressurized to 1 atm argon headspace. Sterile C-, N-, and S-free media (1% PBS, 30 g/L NaCl, 12 g/L MgCl2, and 3 g/L KCl) was prepared anaerobically with deuterated water (20 at. % 2H2O). 20 at. % 2H2O was selected as the highest level of enrichment with little to no effect on the activity of microorganisms in pure culture. Time point 1, time point 2, and autoclaved treatments were prepared for each substrate condition. Time point 1 incubations lasted for six months, while time point 2 and autoclaved treatments were maintained at the in situ incubation temperature for 2.5 years. Due to low levels of activity ascertained from geochemical measurements, all NanoSIMS analyses were conducted on time point 2 and autoclaved samples. Amendments and incubation conditions for the methyl-substrate subset analyzed in this study are provided in this dataset. Equimolar amounts of substrate (30 umol C, 1.5 mM final; 3 umol N, 0.15 mM final) were added across incubation conditions at 50 at. % (Cambridge Isotopes). Hydrogen was added as 5 mL 100% H2 overpressure to incubations (~15% H2 headspace). A full list of the additional incubation conditions prepared onboard are listed in cruise Methods. Alkalinity (34.39 – 9.68 mM) ammonium (2.80 – 1.83 mM) concentrations from formation fluid samples collected onboard exceed concentrations of C and N amendments. Concentrations of methylamine (0.05 mM) and methanol (1 mM) measured from lignite coal also suggest our substrate additions were environmentally relevant. After 30 months of incubation (March 2014) all treatments were sampled for geochemical analyses prior to preparation for NanoSIMS. 3 ml of headspace gas was removed to a vial filled with 0.1 M NaOH for methane analysis. About 1 ml of liquid was filtered through a 0.1 um 13 mm Whatman Polycarbonate Nuclepore Track-Etched Membrane (110405) for DIC analysis. See Supplemental Methods of Trembath-Reichert et al. for detailed description of methane and DIC analyses.

Sample preparation for NanoSIMS analysis
To overcome technical challenges for NanoSIMS analysis of low biomass samples, cell separation and fluorescence-activated cell sorting (FACS) were used to directly concentrate cells in a small analysis area, ~1 to 0.5 sq. mm. NanoSIMS samples were prepared from paraformaldehyde (PFA)-fixed cell separates after 894 days of incubation. Cell preservation, separation, enumeration, and FACS were all conducted in the clean booth and clean room facilities at Kochi Institute for Core Sample Research, JAMSTEC. Half of the solid and half of the liquid portion of each sample were fixed overnight in a solution of 2% paraformaldehyde (PFA), 3 × phosphate buffered saline (PBS). Samples were then subjected to two washes, incubating in 3 × PBS for 6 hrs and then 2 hrs, after each wash respectively. Samples were centrifuged (3500 × g) and supernatant was decanted after each wash. PFA-fixed samples were stored in 50 % ethanol : 3 × PBS. The other half of the sample was preserved in glyTE (70% glycerol, 100mM Tris, 10mM EDTA; Bigelow Single Cell Genomics Center preservation protocol) and frozen by cell alive system (CAS) and stored at -80C. 1 ml liquid and ~1 g sediment chips were subsampled by pipet and sterile cell culture loop, respectively, from the PFA-fixed sample. Cell separation, microscopy, and sorting procedures followed Morono et al., with the following modifications: 1) samples were sonicated (Bioruptor UCD-250, COSMO BIO) in an ice bath for 20 cycles of 30 sec 200 W, 30 sec off, and 2) samples were incubated in hydrofluoric acid post initial sonication, rather than after first density gradient separation. Cell detection limit was determined by no-sample added controls run in parallel with samples. Cells were stained with SYBR Green I (1:40 dilution of SYBR Green in Tris (10 mM) –EDTA (1mM) (TE) and sorted following the flow cytometry protocol of Morono et al. Sorted cells were concentrated directly from the sorter onto NanoSIMS compatible 0.2 um polycarbonate filters coated with indium tin oxide (ITO) as described in Morono et al. and Inagaki et al. ITO coating on polycarbonate membranes (Isopore GTBP02500 Millipore) was prepared by sputtering deposition technique at Astellatech Co. Ltd. (Kanagawa, Japan). Scanning electron microscopy (SEM) of the filters was done on a Zeiss 1550 VP Field Emission Scanning Electron Microscope at the GPS Division Analytical Facility at Caltech and SYBR stained cells were imaged with a BX51 epifluorescence microscope (Olympus, Tokyo, Japan) using 20× (UPlanFL N) dry, 60× (PlanApo N), and 100× (UPlanFL N) oil immersion objectives.