GSM9064854: FAB@MN-2; Homo sapiens; RNA-Seq
HSFs were seeded at a density of 1 × 10^5 cells/well in a 24-well plate and cultured for 24 hours in DMEM containing 10% FBS. Subsequently, the cells were treated with FAB NPs for 4 hours, followed by 10 mins of illumination, and then incubated for another 24 hours. Total RNA was extracted using TRIzol reagent (Invitrogen, USA), and the integrity of the RNA was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA samples were prepared using total RNA as a template. Initially, mRNA was extracted from total RNA using magnetic beads coated with poly-T oligonucleotides. This mRNA was then fragmented under high temperatures in the presence of divalent cations, using a 5X First Strand Synthesis Reaction Buffer. The synthesis of the first cDNA strand was achieved using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). This was followed by the synthesis of the second cDNA strand using DNA Polymerase I and RNase H. Exonuclease and polymerase activities were employed to transform remaining overhangs into blunt ends. Subsequent to the adenylation of the 3' ends of the DNA fragments, adapters containing hairpin loop structures were attached, setting the stage for hybridization. cDNA fragments ranging in size from 370-420 bp were isolated using the AMPure XP system (Beckman Coulter, Beverly, USA). Amplification of these fragments was then carried out using Phusion High-Fidelity DNA Polymerase, along with universal PCR primers and an Index (X) Primer. The PCR products were purified within the AMPure XP system, and the library's quality was evaluated using an Agilent Bioanalyzer 2100 system. Indexed samples were clustered on the cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) according to the manufacturer's guidelines. Post-clustering, the library was sequenced on the Illumina Novaseq platform, producing 150 bp paired-end reads and thereby acquiring the sequence data of the fragments. Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in First Strand Synthesis Reaction Buffer(5X). First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase(RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 370~420 bp in length, the library fragments were purified with AMPure XP system. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.