GSM8956751: Ctrl-1; Mus musculus; RNA-Seq
Cultured cardiac fibroblasts were washed by PBS twice and added with 1 ml of TRIzol reagent on ice, followed by addition of 200 µl of chloroform and vigorous shaking. Each sample was centrifuged at 25,000×g in 4°C for 15 minutes, and the top layer was transferred to a new RNase free tube. Then, 500 µl of isopropanol was added to the tube and  incubate at room temperature for 10 minutes,followed by  centrifugation at 25,000×g in 4°C for 15 minutes.  Supernatant was removed and the RNA pellet was washed by 75% ethanol, and finally dissolved in Rnase-free water. RNA library construction was performed after confirming that the sample RNA integrity numbers were greater than seven using a TapeStation RNA screentape. A library was independently prepared with 1 µg of total RNA from each sample using an Illumina TruSeq Stranded mRNA Sample Prep Kit. As for library kit, TruSeq Stranded mRNA Library Prep Kit was used. As for library protocol, TruSeq Stranded mRNA Reference Guide #1000000040498 v00 was used. The first step in the workflow involved purifying the poly-A containing mRNA molecules using poly-T-attached magnetic beads. Following purification, mRNA was fragmented into small pieces using divalent cations at elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using SuperScript II reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA Polymerase I, RNase H, and dUTP. These cDNA fragments went through an end repair process, the addition of a single 'A' base, and ligation of the adapters. The products were purified and enriched using PCR to create a final cDNA library. The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide, and qualified using the TapeStation D1000 ScreenTape system. Indexed libraries were then submitted to an Illumina NovaSeq6000 (Illumina) and paired-end (2×100 bp) sequencing reads were generated on the Illumina sequencing NovaSeq platform.