Savant spd121p speedvac concentrator

Fatty acids within the lipid extracts were transmethylated to fatty acid methyl esters (FAMEs) by esterification for analysis by GC-MS. The dried lipid extract was transmethylated with an 8% solution of HCl in methanol/water (85:15, v/v) and toluene, and incubated overnight at 45 °C, following which the methanol was evaporated, samples dried using a Savant™ SPD121P SpeedVac™ Concentrator (Thermo Fisher Scientific), and hexane was added, making the solution biphasic. The hexane layer was extracted and transferred to a new glass vial and dried under nitrogen. The dried sample was reconstituted in dichloromethane and analysed by GC-MS using a 6890 GC 5973 N MSD system and 7683 Series Injector, Agilent Technologies, with a ZB-5 column (30 M × 25 mm × 25 mm, Phenomenex), with 1 μL splitless injection, and a GC temperature programme of 70 °C for 10 min, increase in temperature gradient up to 220 °C at 5 °C/min, and 220 °C for 15 min. FAMEs were identified by comparison of retention time and fragmentation pattern, and normalised to the C15:0 as the internal standard, derived from the previously added SPLASH® LIPIDOMIX® Mass Spec Standard (Avanti Polar Lipids: 330707).

The V5, V6, and V7 regions of the 16S rRNA gene were amplified from each sample using the 799F and 1193R primers with Illumina MiSeq adapters and custom pads, linkers, and barcode sequences (61 (link)). The PCR volume was 25 μl: 1 μl of 10× diluted DNA template, a 0.2 μM concentration of each primer, 1× 5PRIME HotMasterMix (5PRIME, Gaithersburg, MD, USA), and 0.8× SBT-PAR additive (5× stock: 750 mM sucrose, 2 mg/mL BSA, 1% Tween-20, 8.5 mM Tris-Cl pH 7.5) (62 (link)). PCR amplification consisted of initial denaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 54.3°C for 40 s, and elongation at 68°C for 40 s, followed by a final elongation at 68°C for 7 min. Each PCR was completed in triplicate, and products were pooled and purified with an equal volume of Axygen AxyPrep Mag PCR Clean-Up bead solution (Corning, Tewksbury, MA, USA). Amplicon concentrations were quantified by fluorimetry (QUANT-iT PicoGreen double-stranded DNA [dsDNA] assay kit; Life Technologies, Carlsbad, CA, USA) and 30 ng or a maximum of 30 μl per sample was pooled for six plates per sequencing run. Primer dimers and mitochondrial amplicons were removed by concentrating each amplicon pool 20× (Savant SPD121P SpeedVac concentrator; Thermo Scientific) and purifying the 300- to 700-bp product with BluePippin (Sage Science, Beverly, MA, USA).