Nanodrop one onec spectrophotometer

RNA immunoprecipitation (RIP) was performed on the C4-2 cell line. The cell was treated with cell lysis buffer. The 10% lysis sample was stored and named “Input”, and 90% was used in immunoprecipitation reactions with anti-ALYREF antibody) and named “IP”, and 10% was incubated with rabbit IgG (Cell Signaling Technology) as a negative control and named “IgG”, respectively. More details were shown in our previous study [21 (link)].
Total RNAs were extracted from the C4-2 cell line using TRIzol Reagent. DNA digestion was carried out after RNA extraction by DNaseI. RNA quality was determined by with Nanodrop™ One/Onec Spectrophotometer (Thermo Fisher Scientific Inc). Qualified RNAs were finally quantified by Qubit3.0 (QubitTM RNA Broad Range Assay kit, Life Technologies.) 2 μg total RNAs were used for stranded RNA sequencing library preparation (KCTM Stranded mRNA Library Prep Kit for Illumina^®, NO. DR08402, Wuhan Seqhealth Co., Ltd. China) following the instruction. PCR products corresponding to 200–500 bps were enriched, quantified, and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.

Pools of four hippocampal tissue samples from the 5XFAD control and 5XFAD UB‐SCG‐51 mice (5 mg/Kg) were extracted using Trizol reagent (Thermo Fisher) and the purity and concentration was evaluated using NanoDrop One/One^C spectrophotometer (Thermo Fisher). The sample quality was assessed using Agilent Bioanalyzer 2100 and sequencing libraries were prepared using the RNA total amount of 5 μg. After QC confirmation, RNA library preparation was performed using TruSeq RNA Sample Prep Kit (Poly A capture). The samples were aligned with the reference genome using Bowtie2, and the gene expression level was estimated using RSEM. Differentially expressed genes were identified using the edgeR program. Genes showing altered expression with p < 0.05 and more than 1.5‐fold changes were considered differentially expressed. KEGG, Gene Ontology, and GSEA were used to perform the enrichment and pathway analysis using Enrichr database. Raw data were deposited at the Gene Expression Omnibus (accession GSE189250), raw fastq files for RNA‐seq on mouse hippocampus 5XFAD (accession GSE189249).

The release assay of IND was carried out using the Spectra-Por Float-A-Lyzer G2 dialysis device (Spectrum Labs, Rancho Dominguez, CA, USA) at room temperature. For the release experiment, 1 mL of the nanoemulsion was introduced into the dyalisis bag and placed in a vessel containing 100 mL of the receptor solution, Hepes buffer, under magnetic stirring. At predetermined time intervals (0, 15, 30, 45, 60, 120, 240, and 1200 min), 50 µL of sample were withdrawn. These samples were extracted according the Bligh and Dyer method, and the organic phase was read in a NanoDrop One/Onec spectrophotometer (Thermo Scientific, Rockford, IL, USA).

Total DNA and RNA were isolated from GCs using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland) and PureLink™ RNA Mini Kit (Invitrogen™, Waltham, MA, USA), respectively, according to the manufacturer’s instructions. Those protocols included the use of spin columns used to isolate high-quality total DNA and RNA, and DNase as treatment to remove genomic DNA from RNA [40 (link)]. After extraction, the samples were stored at −80 °C. The concentration and quality of DNA and RNA were determined using a NanoDrop™ One/OneC Spectrophotometer (ThermoFisher Scientific™, Waltham, MA, USA).

Using the High Pure PCR template preparation kit (Roche, Basel, Switzerland), the genomic DNA of H. pylori was isolated according to the manufacturer’s instructions. DNA quantification and quality control were completed immediately after isolation using a NanoDrop One/OneC Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The DNA samples’ ratio of absorbance at 260/280 nm (OD 1.8–2.0) and 260/230 nm (OD 2–2.2) was considered for DNA sequencing analysis.

RNA extraction was performed using the NucleoSpin® RNA Plus Kit (MACHEREY–NAGEL™) according to the manufacturer’s instructions with minor modifications. The starting sample volume, lysis buffer (LBP) and binding solution (BS) were modified to 200 µl, 300 µl and 120 µl, respectively. Centrifugation (Eppendorf™ 5430) steps were performed at 11,000×g for 2 min instead of 30 s. The RNA was solubilized in 30 µl of RNase-free water. RNA concentration was quantified using a NanoDrop™ One/OneC Spectrophotometer (Thermo Fisher Scientific™, USA).