Rna miniprep kit

RNA was isolated from C57BL/6 and Stat2^−/− mouse lungs using the Agilent RNA miniprep kit. RNA integrity was determined using an Agilent 2100 bio analyzer. mRNA was purified by using Sera-Mag Oligo(dT) Beads, fragmented with magnesium-catalyzed hydrolysis, and reverse transcribed into cDNA using random primers (Superscript II; Invitrogen). Then, cDNA underwent end repair with T4 DNA polymerase and Klenow DNA polymerase, followed by the addition of “A” bases to the 3′ end and ligation to adaptor oligonucleotides. Products from the ligation were run on a 2% agarose gel. A gel slice consisting of the 200 bp region (±25 bp) was excised and used as a template for PCR amplification. The final PCR product was purified, denatured with 2 N NaOH, and diluted to 10–12 pM prior to cluster amplification on a single-read flow cell v4, as outlined in the Single-Read Cluster Generation Kit v4 (Illumina). The flow cell was sequenced on an Illumina Genome Analyzer II. The data were analyzed as previously described (24 (link)). Full sequencing data has been uploaded to the National Center for Biotechnology Information Gene Expression Omnibus, GSE119029.

2-Hydroxymelatonin (2(OH)Mel), 6-hydroxymelatonin (6(OH)Mel), bovine serum albumin (BSA), ethanol (EtOH), glucose, melatonin, and sodium pyruvate were purchased from Sigma (St. Louis, MO, USA). AFMK and GlutaMAX™ Supplements were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Thermo Fisher Scientific (Waltham, MA, USA), respectively. Other reagents were supplied as follows: EpiGRO™ Human Epidermal Keratinocyte Complete Culture Media Kit (Millipore Merck KGaA, Darmstadt, Germany); RNA Miniprep Kit (Agilent Technologies, Santa Clara, CA, USA), high capacity cDNA Reverse Transcription Kit (Applied Biosystems), DyNamo Flash SYBR Green qPCR Kit (Thermo Scientific, Waltham, MA, USA), KAPA SYBR^® FAST qPCR Kit (Kapa Biosystems, Wilmington, MA, USA), the Cell Mito Stress Test media was supplemented with 2 mM GlutaMAX™ Supplement (L-alanyl-L-glutamine dipeptide in NaCl), Proteome Profiler Array kit (R&D Systems, Minneapolis, MN, USA), lactate colorimetric assay kit (Cell Biolabs, Inc. San Diego, CA, USA), TeloTAGGG Telomerase Elisa assay (Roche, Basel, Switzerland), and a set of human primers for real-time PCR (Eurofins Genomics, Ebersberg, Germany).

Total RNA was isolated from cultured astrocytes (1×10⁵) using RNA mini‐prep kit (Agilent Technologies, Santa Clara, CA, USA). First‐strand cDNA synthesis was carried out with reverse transcriptase Superscript TMIII (Invitrogen, Barcelona, Spain) using random primers. Specific primers for NOX1‐NOX4 and DUOX1, DUOX2 were obtained from a previous study (Reinehr et al., 2007). Real‐time quantitative PCRs were carried out with 25 ng of reverse‐transcribed RNA and 300 nM of primers diluted in SYBRGreen PCR master mix reagent (Invitrogen, Barcelona, Spain). Relative levels of expression of target genes were calculated by means of a normalization factor, based on the geometric mean of multiple internal control genes and calculated by software.