Nanodrop nd 1000 system

IgG was purified from frozen serum samples using Magne Protein G Beads (Promega, Madison, WI, USA) according to manufacturer’s instructions. Briefly, 50 μl beads were incubated with 50 μl pre-centrifuged thawed serum samples for 30–60 min at room temperature while shaking. Supernatant was removed via magnetic separation of the beads, which were subsequently washed multiple times. Purified IgG was eluted from the beads, and the purified IgG quality and concentration was obtained using the NanoDrop^® ND-1000 system (Thermo Fisher Scientific). Purified IgG fractions were stored at 4°C until further usage.

The total RNA was extracted from the primary cultured neurons using TRIzol reagent (Invitrogen, Carlsbad, CA, USA; Cat. No. 15596018) according to the manufacturer’s protocol. A NanoDrop ND-1000 system (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the RNA concentration in each sample. The OD260/OD280 value ratio was assessed as an index of the RNA purity, and OD260/OD280 values ranging from 1.8 to 2.1 indicated that the RNA met the qualifications for purity. The RNA integrity was measured using denaturing agarose gel electrophoresis.

Snap frozen brain tissue (frontal cortex and basal ganglia) (~ 30 mg) was disrupted using a needle and syringe. Total RNA was extracted using a Qiagen RNeasy (Scientifix, VIC, AUS), according to the manufacturer's instructions. RNA yield and quality were determined using a NanoDrop spectrophotometer (NanoDrop ND-1000 system, Thermo Fischer Scientific). A 260/280 ratio of ~ 2.0, and a 260/230 ratio 2 > 2.2 was accepted for RNA as per Thermo Scientific Technical Bulletin T042.

According to the standard protocol, DNA was extracted from the fecal samples using QIAamp DNA Stool Mini Kit (Qiagen, Dusseldorf, Germany). A NanoDrop ND-1000 system (Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the DNA concentration. Universal primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) were used to amplify the V3–V4 regions of 16S rDNA. The polymerase chain reaction (PCR) products were quantified and purified using a QuantiFluor™ fluorometer (Promega Biotech, Madison, WI, USA). Negative controls included no templates for DNA extraction or PCR amplification. The PCR products were mixed in equidensity ratios. The mixture of the PCR products was purified with a Gel Extraction Kit (Qiagen). Sequencing libraries were generated using the TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) following manufacturer’s instructions, with the addition of index codes. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific) and Bioanalyzer 2100 system (Agilent). Finally, the library was sequenced on a HiSeq2500 platform (Illumina), and 250 bp paired-end reads were generated.

RNA isolation was performed using the Nucleospin RNA II kit (Macherey-Nagel). The concentration of RNA was measured using a NanoDrop ND-1000 system (Thermo Fisher Scientific). RNA quality and integrity were assessed by the TapeStation 2200 system (Agilent Technologies). For qRT–PCR expression analysis, the RNA was reverse transcribed using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative expression analysis was performed using the QuantStudio 5 Real-Time PCR instrument and predesigned TaqMan gene expression assays (Thermo Fisher Scientific): C14ORF105 (Taqman probe Hs00216847_m1). GAPDH expression was used as an internal control. Relative quantification was performed according to the ΔΔCT method^{88 (link)}.

Total RNA was extracted from PBMCs with TRIzol reagent (Invitrogen, Carlsbad, CA). The purity and concentration of total RNA samples were determined with a NanoDrop ND-1000 system (Thermo Fisher Scientific, Waltham, MA). The optical density (OD) 260/280 absorbance ratios of all the samples were between 1.8 and 2.0.

Total RNA was extracted from primary cultured neurons using TRIzol reagent (Invitrogen, Carlsbad, CA, USA; Cat. No. 15596018) according to the manufacturer’s protocol. A NanoDrop ND-1000 system (Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the RNA concentration in each sample. The OD260/OD280 ratio was assessed as an index of RNA purity, and samples with OD260/OD280 values ranging from 1.8 to 2.1 met the qualifications for purity. RNA integrity was evaluated using denaturing agarose gel electrophoresis.