Primer sets

SYBR Green was used to compare the gene profile levels under different processing conditions following normoxia and hypoxia. cDNA was first synthesized from mRNA using a PrimeScript^TM RT reagent kit according to the manufacturer's protocol. The resulting cDNA was standardized across the samples depending on the sample concentration. Then, the gene profile was amplified over the course of 30 cycles and analyzed by the ∆∆CT method. Primer sets predesigned for the following genes were purchased from Invitrogen. The sequences are listed in Table S5 and S6.

The total RNA of microglia cells was extracted using Quick-RNA MiniPrep Kit (Zymo Research) following the manufacturer’s instructions. cDNA was synthesized from 1 μg of total RNA using the reverse transcriptase Improm-II enzyme (Promega). Real-time quantitative PCR (RT-qPCR) was performed with the master mix FastStart Universal SYBR Green Master (ROX, Roche) in a StepOne Real-Time PCR System (Applied Biosystems). Primers used were as follows: TNF-α forward: 5’-ATGGCCTCCCTCTCATCAGT-3’, reverse: 5’-TTTGCTACGACGTGGGCTAC-3’; IL-1β forward: 5’-GCCACCTTTTGACAGTGATGAG-3’, reverse: 5’-GACAGCCCAGGTCAAAGGTT-3’; IL-6 forward: 5’-AGACAAAGCCAGAGTCCTTCAG-3’, reverse: 5’-GAGCATTGGAAATTGGGGTAGG-3’; iNOS forward: 5’-CAGCTGGGCTGTACAAACCTT-3’, reverse: 5’-CATTGGAAGTGAAGCGTTTCG-3’; β-actin forward: 5’-AACAGTCCGCCTAGAAGCAC-3’, reverse: 5’-CGTTGACATCCGTAAAGACC-3’. The amplification cycle was the following: 10 min at 95°C, 40 cycles at 95°C for 15 s, 60°C for 30 s, and 72°C for 60 s. All primer sets (Invitrogen) yielded a single amplification product by melting curve analysis. The fold change (relative expression) in gene expression was calculated using the relative quantitation method (2^−ΔΔCt). Relative expression levels were normalized to the expression of β-actin and plotted in a log₂ scale.

The entire mtGenome was amplified by long PCR using primers that generate two amplicons approximately 8.5 kb in length in separate reactions as described by Gunnarsdóttir et al. [33 (link)]. The PCR included SequalPrep^TM 10× Reaction Buffer (Life Technologies), SequalPrep^TM 10× Enhancer B (Life Technologies), SequalPrep^TM long polymerase (5U/µl) (Life Technologies), DMSO (Life Technologies), primer sets (Life Technologies), DNase-free water, and 5 ng of total genomic DNA according to the manufacturer’s protocol. The amplification conditions were 2 min at 94 °C for polymerase activation, 30 cycles of 10 s at 94 °C for denaturation, 30 s at 60 °C for annealing, 8 min at 68 °C for extension; followed by a final extension of 5 min at 72 °C. The two amplicons were pooled in equimolar amounts (i.e., 50 ng each). The PCR amplicons were enzymatically fragmented using Ion Shear™ Plus Reagents (Life Technologies) and for one experiment by physical shearing with the Covaris system (Covaris, Woburn, MA) following the manufacturer’s protocol. Ion adapters and barcodes were ligated to the fragmented amplicons using the Ion Plus Fragment Library and Ion Xpress™ Barcode Adapters Kits (Life Technologies). The library was size-selected at 315 bp with the Pippin Prep™ instrument (Sage Science, Beverly, MA).