Tri reagent protocol

We extracted RNA using the TRI Reagent Protocol (Sigma-Aldrich, USA) from samples preserved in RNAlater (Qiagen, USA). Any remaining genomic DNA was removed using the TURBO DNA-free Kit (Invitrogen, USA). Each treatment included five biological replicates, with each replicate comprised of 35 clonal Daphnia individuals. Extracted RNA was stored in -80°C until sequencing. Quality of the isolated RNA was assessed using RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) and mRNA-seq libraries were constructed using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following manufacturer’s recommendations. A total amount of 1 μg RNA per replicate was used as input material for RNA-seq library preparations. Samples that did not reach 1 μg RNA were pooled with a biological replicate grown under the same conditions, a process described by Takele Assefa et al. [34 (link)], leaving 18 replicates from Blue Lake and 16 replicates from Gardisky Lake. Index codes were added to attribute sequences to each sample and all 34 libraries were clustered on a cBot System using the PE Cluster kit cBot-HS (Illumina). After generating the clusters, libraries were sequenced using the Illumina HiSeq 2000 platform and 150 bp paired-end reads were generated.

Percoll-enriched microglia were sorted using a Becton-Dickinson FACSAria III cell sorter at the OSUCCC Analytical Cytometry facility. Microglia were identified by CD11b⁺/CD45^low expression. Cells were pelleted and lysed in Arcturus PicoPure Extraction Buffer immediately after sorting. The Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) was used to purify and concentrate total RNA from sorted microglia. During RNA isolation, samples were treated with on-column DNase digestion for 15 min at 23 °C to eliminate contaminating genomic DNA. A 1-mm coronal brain section was also collected from each brain and snap frozen in liquid nitrogen. RNA was isolated using the Tri-Reagent protocol (Sigma-Aldrich). RNA quality and integrity was determined using the Agilent 2200 TapeStation assay (Agilent Technologies). RNA-Seq was performed on sorted microglia and brain section (Bregma - 1.5 mm) RNA at the Hussman Institute for Human Genomics Sequencing Core Facility (University of Miami, Miami, FL). Briefly, RNA-Seq libraries were prepared using the Ovation SoLo RNA-Seq System with AnyDeplete rRNA to remove rRNA and other abundant transcripts according to the manufacturer’s recommendation (Nugen). RNA-Seq libraries were run on an Illumina NextSeq 500 sequencing instrument according to the protocols described by the manufacturer.

Yeast samples were grown in 20 ml YPD medium to mid-log phase (0.8 OD600 value). RNA was extracted from 10⁷ yeast cells by acidic phenol method using TRI Reagent Protocol (Sigma-Aldrich Co). The RNA samples were concentrated by the NucleoSpin RNA Plant Kit (Macherey-Nagel), according to the manufacturer's instructions. A total of 500 ng RNA was used as a template to prepare cDNA using the Maxima First Strand cDNA Synthesis kit (Thermo Scientific). Reactions without template were set up to detect contaminations of the reagents used in the cDNA synthesis. qPCR reactions were set up in 20 µl volume, using the following templates: no template control, 10 ng non-transcribed RNA and cDNA transcribed from 10 ng RNA. The qPCR reactions were run in a Bioer LineK Gene device, using 2× Maxima SYBR Green qPCR Master Mix (Thermo Scientific). All samples had three technical replicates. Gene expression was determined in arbitrary units using a standard curve fitted on triplicates of a four-step 10-fold dilution series. OLE1 expression level was determined relative to TUB1 expression level as an internal control. All control reactions, not treated with reverse transcriptase or not having template, gave Ct values at least 10 cycles higher than the corresponding samples.

Mice were sacrificed by CO₂ asphyxiation and the hippocampus was dissected and snap frozen in liquid nitrogen (− 196 °C). Hippocampal RNA was isolated using the Tri-Reagent protocol (Sigma-Aldrich). RNA quality and integrity was determined using the Agilent 2200 TapeStation assay (Agilent Technologies). nCounter analysis (NanoString Technologies) was performed by the OSU Comprehensive Cancer Center (OSUCCC) Genomics Shared Resource facility (The Ohio State University, Columbus, OH) using the Mouse Inflammation v2 Panel for 248 inflammation-related mouse genes, 20 custom genes, and 6 internal reference controls. Copy numbers were normalized using DESeq2 Bioconductor package in R [41 (link)].

RNA isolation was carried out according to the Tri Reagent® Protocol (Sigma-Aldrich) with some modifications: homogenization of the samples was performed in Tri-reagent (Sigma-Aldrich) using 1.4 mm zirconium oxide beads (Precellys 24) and a TissueLyser LT (Qiagen) for 7 min at 50 Hz. The RNA was extracted as described before [24 (link)]. The purified RNA was dissolved in RNase free water and quantified using a Nanodrop 2000 Spectrophotometer (NanoDrop Products). RNA samples were either frozen at—80°C or used directly for the production of complementary DNA (cDNA). For cDNA synthesis, the samples were DNase treated according to the manufacturer´s protocol (Turbo DNase free^TM kit, Ambion Foster City, CA). RNA (200 ng) was reverse transcribed to cDNA using the Affinity Script cDNA Kit (Agilent) according to the manufacturer’s instructions. Before storage at—20°C the cDNA was diluted ten times for quantification of transcript levels described in (2.9).