Transcriptional profile of monocytes under high glucose in Stanford A acute aortic dissection
Transcriptional profile of monocytes under high glucose in Stanford A acute aortic dissection
Using magnetic beads to isolate CD14⁺ monocytes from  Stanford type-A acute aortic dissection (ATAAD) patients.CD14⁺ monocytes were cultured in RPMI-1640 medium with 10% FBS and treated with 5.5 mM or 30 mM glucose for 24 hours at 37°C, 5% CO₂.After the incubation, cells were harvested for RNA extraction and subsequent RNA sequencing. CD14⁺ monocytes were obtained from patients diagnosed with Stanford type-A acute aortic dissection (ATAAD). All procedures were approved by the Second XiangYa Hospital Ethics Committee, and written informed consent was obtained from all participants.  CD14⁺ monocytes were subsequently purified CD14 MicroBeads and magnetic-activated cell sorting (MACS) according to the manufacturer's instructions. The purity of the isolated CD14⁺ monocytes was confirmed by flow cytometry to be >95%. CD14⁺ monocytes were cultured in RPMI-1640 medium with 10% FBS and treated with 5.5 mM or 30 mM glucose for 24 hours at 37°C, 5% CO₂. CD14⁺ monocytes were treated with normal glucose (5.5 mM; n=4) or high glucose (30 mM; n=4) medium for 24 hours. Base calling and demultiplexing of raw sequencing data (BCL files) were performed using Illumina's `bcl2fastq` (version 2.20) software. Adapter sequences were automatically trimmed during the process. The output was converted to demultiplexed FASTQ files for each sample. Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA,USA) according  to the manufacturer's protocol. After enriching eukaryotic mRNA with poly(A) tails using oligo(dT)-coupled magnetic beads, the mRNA is fragmented with a fragmentation buffer. Using this fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA is synthesized in an M-MuLV Reverse Transcriptase system. The RNA strand is then degraded by RNase H, and the second strand of cDNA is synthesized in a DNA Polymerase I system using dNTPs. The purified double-stranded cDNA undergoes end repair, poly(A) tailing, and sequencing adapter ligation. cDNA fragments of approximately 200 bp are selected using AMPure XP beads, followed by PCR amplification. The PCR products are purified again with AMPure XP beads to finally obtain the sequencing library.