GSM8511862: BV2 cells,control1(6h); Mus musculus; RNA-Seq
For RNA extraction, BV2 microglial cells were subjected to glucose deprivation followed by RNA isolation using the TRIzol reagent (Invitrogen). Cells were lysed directly in the culture dish, and total RNA was extracted according to the manufacturer's instructions. The RNA integrity was assessed using an Agilent 2100 Bioanalyzer, ensuring an RNA integrity number (RIN) greater than 8.0. Library preparation for sequencing was conducted using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. Following manufacturer's protocols, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out under elevated temperature in NEBNext First Strand Synthesis Reaction Buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase. Subsequently, second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. Library fragments were purified with the AMPure XP system and enriched with PCR to create the final cDNA library. The library strategy used is RNA-Seq, which involves direct sequencing of cDNA fragments. This method is utilized to capture a comprehensive snapshot of the transcriptomic profile of glucose-deprived BV2 microglial cells, focusing on both coding and non-coding RNA to investigate cellular responses to glucose deprivation at the molecular level.