Qubit3.0 with qubit rna broad range assay kit

A stranded RNA sequencing library was prepared according to manufacturer’s instructions by using KCTM Stranded mRNA Library Prep Kit (Catalog No. DR08402, Wuhan Seqhealth Co., Ltd., Wuhan, China) (18 (link)). Briefly, the mRNA was enriched by magnetic beads and broken into short fragments by fragment buffer. A random primer was used to synthesize a single strand of cDNA using the mRNA fragment as a template. After synthesizing and purifying double stranded cDNA, the terminus of the cDNA was repaired with base and added with sequencing adaptor. The fragment was caught by magnetic beads and amplified by T100 Thermal Cycler (BIO-RAD, United States). Qubit 3.0 with Qubit RNA Broad Range Assay kit (Life Technologies, Q10210) is used to quantify the cDNA library. Finally, the different cDNA library corresponding to 200–500 bps was sequenced on Illumina Novaseq 6000 sequencer (Illumina Inc., San Diego, CA, United States) with a pair-end 150 bp (PE150) model at Seqhealth Technology Co., LTD (Wuhan, China). The RNA-seq data were obtained based on two independent biological replicates. Each group includes two samples and all the samples were repeated for two times.

K562/G01 cells were cultured in normal (control) or cysteine depletion (treat) condition for 24 h. The total RNA of the above two groups was isolated using TRIzol (CWBIO, #CW0580S) following the manufacturer's instructions. Qualified RNAs were finally quantified by Qubit3.0 with Qubit™ RNA Broad Range Assay kit (Life Technologies, USA) and used for stranded RNA sequencing library preparation using KC™ Stranded mRNA Library Prep Kit for Illumina® (Wuhan Seqhealth Inc., China). PCR products corresponding to 200-500 bp were enriched, quantified, and finally sequenced on Hiseq X10 sequencer (Illumina, USA).

Mice of the four groups (PKAi_CD, PKAi_HFD, Control_CD and Control_HFD) were raised as described above. Then, 3 mice of each group were sacrificed and their liver tissues were harvested for RNA isolation using the TRIzol (Invitrogen, US) as described in the instruction. RNA quality was determined by examining A260/A280 with a Nanodrop™ OneCspectrophotometer (Thermo Fisher Scientific Inc., US). RNA Integrity was confirmed by 1.5% agarose gel electrophoresis. Qualified RNAs were finally quantified by Qubit3.0 with Qubit™ RNA Broad Range Assay kit (Life Technologies, US) and used for stranded RNA sequencing library preparation using KC™ Stranded mRNA Library Prep Kit for Illumina® (Wuhan Seqhealth Inc., China). PCR products corresponding to 200–500 bp were enriched, quantified and finally sequenced on Hiseq X10 sequencer (Illumina, US).

Total RNA was extracted from ADSC-NVs and ADSC-EVs using SeraMir RNA purification kit (System Biosciences, RA808A-1) in accordance with the manufacturer’s protocol. RNA quality was examined using Nanodrop™ One C spectrophotometer, and qualified RNAs were subsequently quantified using Qubit3.0 with Qubit™ RNA Broad Range Assay kit (Life Technologies, Q10210). miRNA sequencing library was obtained using Small RNA Sample Pre Kit (TruSeq, Illumina) following the manufacturer’s instructions. Small RNAs were reverse transcribed and amplified through PCR, followed by sequencing of PCR products using Illumina NovaSeq 6000 platform. For miRNA-seq data analysis, raw reads were filtered using fastx_toolkit (version: 0.0.13.2) and adaptor sequences were removed using cutadapt (version: 1.15). The target genes of miRNA in ADSC-NVs and ADSC-EVs were predicted using miRanda (http://www.microrna.org/). Finally, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the predicted target genes were performed using DAVID Bioinformatics.

Total RNA was extracted from skin tissue (samples same as transcriptome sequencing) using TRIzol Reagent (Invitrogen). After RNA extraction, DNaseI was used to remove residual gDNA. RNA quality was examined by Nanodrop onec spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was visualized by 1.0% agarose gel electrophoresis. Qualified RNA was finally quantified by Qubit3.0 with Qubit RNA Broad Range Assay kit (Life Technologies, Carlsbad, CA, USA). Subsequently, 50 μg total RNA of each sample was calculated for polyadenylated RNA enrichment by using VAHTS mRNA Capture Beads (VAHTS). This was followed by 20 mM ZnCl₂ added to the RNA tube, mixed well, and incubated at 95 °C for 5–10 min until the RNA fragments were mainly distributed in 100~200 nt. Then, 10% RNA fragments were saved as “Input” and the rest was used for m6A immunoprecipitation (IP). The specific anti-m6A antibody (Synaptic Systems) was applied for m6A IP. After that, the enriched RNA was extracted by using TRIzol Reagent (Invitrogen). The stranded RNA sequencing library was constructed by KC-Digital Stranded mRNA Library Prep Kit for Illumina (Seqhealth), as per the manufacturer’s instruction. The library products of 200–500 nt were enriched and quantified, and finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.