Rnase free dnase 1

C. jejuni 11168 wild type, ΔpseC and ΔpseF mutant cells were harvested from overnight NZCYM plate cultures, pelleted and washed once in NZCYM broth and set to an OD₆₀₀ of 0.05 (2 × 10⁸ colony forming units per mL (CFU/mL)) in 20 mL NZCYM broth in 125-mL Erlenmeyer flasks, each containing a 1-inch sterile magnetic stir-bar. Cells were grown under microaerobic conditions and magnetically stirred at 200 rpm. After 4.5 h incubation (mid-log phase, cell counts were approximately 5 × 10⁸ CFU/mL), the entire contents of each flask was transferred to a pre-prepared tube containing 2.6 mL (0.1 volume) ice cold 10% buffered phenol in 100% ethanol to stabilize RNA followed by immediate mixing and storage on ice until all samples were collected [37 (link)]. RNA was extracted from each sample using a hot phenol method [37 (link)]. RNA samples were sequentially DNAse-treated (37 °C for 30 min) using RNAse-free DNAse I (Epicentre, Madison, WI, USA) and cleaned using the Zymo RNA Clean & Concentrator (Zymo Research, Irvine, CA, USA). PCR was used to confirm the absence of residual DNA. Total RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA was stored at −80 °C until further use. Samples were extracted in biological triplicate.

Cells were harvested from overnight NZCYM plate cultures, pelleted and washed once in NZCYM broth and set to an OD₆₀₀ of 0.05 (2 × 10⁸ CFU/mL) in 20 mL NZCYM broth in 125-mL Erlenmeyer flasks. Cells were grown under microaerobic conditions with agitation at 200 rpm. After 4.5 h incubation, CC-FlaGrab was added to a final concentration of 25 μg/mL. As negative controls, 6 mL HEPES buffer or 6 mL NC-FlaGrab were used. Flasks were incubated 30 min before the entire contents of each was harvested and the RNA stabilized using 0.1 volumes of ice cold 10% buffered phenol in 100% ethanol (Palyada et al., 2004 (link)). Total RNA was extracted from each sample using a hot phenol method as previously described (Palyada et al., 2004 (link)). Genomic DNA was removed from the samples using RNAse-free DNAse I (Epicenter) (37°C for 30 min) and cleaned using the Zymo RNA Clean & Concentrator. PCR was used to confirm the absence of residual DNA and the DNase treatment was repeated until the absence of genomic DNA was confirmed. Total RNA quality was assessed using an Agilent Bioanalyzer and RNA was stored at −80°C until further use. This experiment was done in biological triplicate, with the exception of the NC-FlaGrab control, which was done only once.

A total of 1 mL of plasma was cleared of cellular debris by centrifugation at 3,000 g for 15 minutes at 4°C. The RNA was then isolated using the Qiagen exoRNeasy Serum/Plasma Midi Kit (Qiagen Germantown, MD). The RNA was then digested with RNase-free DNase I (Epicentre, Madison, WI) and re-purified on RNeasy MinElute columns (Qiagen Germantown, MD).

Total RNA was extracted from SVZ-derived NPCs using RNeasy Mini Kit (Qiagen), digested with RNAse-free DNAse I (Epicentre), and purified using RNeasy MinElute columns (Qiagen). cDNA libraries were constructed from DNA-free total RNA (50 ng) using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies), followed by loading onto an Illumina NextSeq 500 v2.5 flow cell cartridge. The libraries were extended, and bridge amplified to create sequence clusters and sequenced with 76 nt paired-end reads plus 8 nt single-index reads using the Illumina NextSeq 500 High Output sequencing reagent kit v2 controlled by the NextSeq Control Software version 2.2.0.4. DESeq2 was employed to examine the effect of METH, EcoHIV, and the interaction of METH and EcoHIV on gene expression level [21 (link)]. Library preparation, sequencing, and generating FASTQ files were performed by Ocean Ridge Biosciences (http://www.oceanridgebio.com/) [22 (link)].

For RNA extractions, gonadal fat pads and hippocampi were homogenized using TRIzol reagent (Invitrogen), following the manufacturer’s protocol. The RNA pellet was treated with RNase-free DNase I (Epicentre) for 30 min at 37°C, and a phenol/chloroform extraction was performed to isolate RNA. The iScript cDNA synthesis system (Bio-Rad) was used to reverse transcribe cDNA from 1 μg of purified RNA. Real-time quantitative PCR was performed on the resulting cDNA using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) and a Bio-Rad CFX Connect Thermocycler. All measurements were performed in duplicates. Quantification of PCR products was conducted by normalizing with a combination of corresponding hypoxanthine-guanine phosphoribosyltransferase (HPRT) and succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial (SDHA) expression levels from the gonadal fat samples, and with β-actin expression levels from hippocampus, using the ΔΔ-CT method to obtain relative mRNA levels. Gonadal fat was probed for levels of cluster of differentiation factor 68 (CD68) and EGF-like module-containing mucin-like hormone receptor-like 1 (F4/80), while hippocampus was probed for β-secretase 1 (BACE1), neprilysin, insulin-degrading enzyme (IDE), CD68, glial fibrillary acidic protein (GFAP), and cluster of differentiation factor 74 (CD74). Primer pair sequences are shown in Table 1.

For transcript level analysis, RNA purified from rat kidneys was treated with RNase-free DNase I, 1 U per 10 µg of RNA in 20 µl reactions, (Epicentre Technologies, Madison, WI) for 15 min at 37°C, deproteinized with phenol/chloroform mixture and precipitated with ethanol. One microgram of DNA-free RNA was reverse transcribed by Superscript II (100 U, Invitrogen, Gaithersburg, MD.) with random hexanucleotide primer mixture (1µM) in 10-µl final volume for 1 hr at 42°C. Reaction was stopped by mixing with 90µl TE (Tris-HCl 10mM, pH8.0, EDTA 1mM) and incubation at 95ºC for 5 min. RT mixtures were further diluted ten times with TE and analyzed by real time PCR with gene-specific sets of primers. Transcript levels were normalized to Gapdh mRNA levels.