Dataset: Metagenomic sequences: IODP Site 1244
Deployment: JRES-204

DNA extraction and purification

DNA was extracted, in duplicate, from 8 to 20 g of of “Mobio” IODP sediment samples previously frozen at -80C  using a Powersoil total RNA Isolation Kit with the DNA Elution Accessory Kit (MO-BIO Laboratories, Solana Beach, CA, USA) following the manufacturer protocol but without beads. Approximately 2 grams of sediments were utilized for each extraction, and DNA pellets from each depth were pooled together. DNA concentrations were measured using a Qubit 2.0 fluorometer with dsDNA High Sensitivity reagents (Invitrogen, Grand Island, NY, USA). The DNA from the extractions that yielded robust 16S rRNA and metagenomic sequences ranged from 3.75-15 ng of DNA per gram of sediments.

Multiple Displacement Amplification (MDA)

DNA was amplified using a REPLI-g Single Cell Kit (Qiagen, Germantown, MD, USA). Tubes, tube caps and reverse transcription-PCR grade water (Ambion, Grand Island, NY, USA) were UV-treated for 15 min in a PCR laminar flow hood. Quantitative PCR showed that the negative control began amplifying after 5 hr of incubation at 30°C, and therefore, the 30°C incubation step was shortened to 5 hr using a Bio-Rad C1000 Touch thermal cycler (Bio-Rad, Hercules, CA, USA). DNA concentrations were measured in duplicate on a Qubit 2.0 with dsDNA High Sensitivity reagents (Invitrogen, Grand Island, NY, USA).

Illumina library preparation and sequencing

Two micrograms of MDA DNA were used to generate genome libraries using a TruSeq DNA PCR-Free Kit following the manufacturer’s protocol (Illumina, San Diego, CA, USA). The resulting libraries were sequenced using a Rapid-Run on an Illumina HiSeq 2500 (Center for Integrative Genomics, Georgia Institute of Technology) to obtain 100 bp paired-end reads