title: Microbiome of algal cultivation isolated from Brant Lake sediment core
design: Cores were collected using a custom-built box corer from a 4-meter cataraft.  The sediment samples were obtained from the deepest areas of each lake, which were located with the aid of bathymetric maps and depth sounders. The sediments were extruded vertically out of the box corer immediately after collection at the shore and in the shade to avoid exposure to sunlight. At Moreau Lake, the sediments were extruded in 1-cm intervals from the surface of the core down to 18 cm, then they were extruded in 2-cm increments to the bottom of the core at 38 cm. At Brant Lake, sediments were extruded every 1 cm from the surface to the bottom of the core at ?? cm. The polybuterate boxes and stainless-steel trays used to subsample each core were cleaned with a lint wiper, soaked in a bleach solution, soaked in a sodium thiosulfate solution, and then rinsed with DI water to prevent contamination between layers. Subsamples for genetic analysis were taken from the center of each sediment layer to prevent possible edge effect contamination.  Sediment core samples were chilled and shipped overnight on ice to the Michigan Bacteriological Research Laboratory (MI-BaRL; Lansing, MI) for cyanobacterial cultivation and molecular processing. Approximately 1 gram from each sediment core was removed and split evenly (0.3 g each) between triplicate PowerSoil Pro Bead Tubes (Qiagen; Germantown, MD) and frozen directly.  A total of 1.5 to 2 g of bed was added to sterile polypropylene tubes containing 35 mL of BG-11 media.  All tubes were inverted and gently vortexed to allow media to come into contact with all of the sample.  Tube caps were loosened to allow for an aerobic environment, and tubes were added to incubated at 20C on a 12-hour diel cycle for six weeks.  Following incubation, visible growth was recorded, and all samples were processed for subsequent DNA extraction and culture preservation.  A total of 20 mL of culture was removed and transferred to a sterile amber polypropylene tube for culture preservation.  The remaining cultures were filtered through a 0.22 m filter that was then placed in a sterile microcentrifuge tube and frozen until subsequent DNA extraction.  Approximately 1 gram of sediment from the culture was split between triplicate PowerSoil Pro Bead Tubes and frozen directly.   DNA was subsequently extracted from sediment cores and culture preservations in triplicate using the Qiagen DNeasy PowerSoil Pro Kit according to manufacturers instructions.  DNA from the triplicate extractions were combined with ethanol precipitation.  All DNA concentrations were quantitated with a Quantus fluorometer (Promega; Madison, WI) using the Promega QuantiFluor dsDNA system. Genomic DNA was delivered frozen to the Michigan State University Research Support Technology Facility Genomics Core (MSU RTSF; East Lansing, MI) for 16S rDNA amplicon sequencing on the Illumina MiSeq sequencing platform (Illumina; San Diego, CA for library preparation and sequencing.  The Bacteria 16S rRNA V4 hypervariable region was amplified with dual indexed, Illumina compatible primers (515f/806r) following the protocol described by Kozich et al., 2013. Following PCR, all amplified products were batch normalized using an Invitrogen (Carlsbad, CA) SequalPrep DNA Normalization plate and the products recovered from this plate pooled. Pools were concentrated and cleaned using a QIAquick Spin column and AMPure XP magnetic beads (Beckman Coulter; Indianapolis, IN). The pool was then checked for quality and quantified using a combination of the Qubit dsDNA HS (Invitrogen; Carlsbad, CA), Agilent 4200 TapeStation HS DNA1000 (Agilent; Santa Clara, CA) and Invitrogen Collibri Illumina Library Quantification qPCR assays (ThermoFisher; Waltham, MA). Each pool was sequenced using an Illumina MiSeq v2 standard flow cell in a 2x250 bp paired end format using a v2 500 cycle reagent cartridge. Custom sequencing and index primers complementary to the 515f/806r target specific sequences were added to appropriate wells of the reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v1.18.54 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0.