title: GSM8316429: HTMCs, DMSO-3; Homo sapiens; RNA-Seq
library_construction_protocol: After 7 days of culture in the presence or absence of 100 nM dexamethasone, to extract total RNA from HTMCs, begin by lysing the cells with 1 ml of TRIzol Reagent per well of a 6-well plate, ensuring complete lysis by vortexing for 15 seconds. Add 0.2 ml of chloroform per 1 ml of TRIzol, shake vigorously for 15 seconds, and let it sit at room temperature for 2-3 minutes. Centrifuge at 12,000 x g for 15 minutes at 4°C, then carefully transfer the upper aqueous phase to a new tube. Precipitate RNA by adding an equal volume of isopropanol, mix gently, and let it stand at room temperature for 10 minutes. Centrifuge at 12,000 x g for 10 minutes at 4°C, discard the supernatant, and wash the RNA pellet with 1 ml of 75% ethanol. Centrifuge at 7,500 x g for 5 minutes at 4°C, discard the supernatant, and air-dry the pellet for a few minutes. Dissolve the RNA pellet in RNase-free water and incubate at 55-60°C for 10 minutes to ensure complete dissolution. After the sample is qualified, the sample library is constructed. The specific mRNA capture and library construction process is as follows: Most eukaryotic mRNAs have a polyA structure. Oligo (DT) magnetic beads are used to capture mRNAs with a polyA structure; The fragmented mRNA is used as a template and random oligonucleotides are used as primers to synthesize the first cDNA chain in the M-MuLV reverse transcriptase system; The RNA chain is degraded with RNaseH, and the second cDNA chain is synthesized with dNTPs as raw materials under the DNA polymerase I system; The double-stranded cDNA is purified, and the end is repaired, the A tail is added, and the sequencing adapter is connected; The cDNA of about 200bp is screened with AMPure XP beads, PCR amplification is performed, and the PCR product is purified again with AMPure XP beads to finally obtain the sequencing library.