GSM8413268: LEG92, Passage 6, Paclitaxel (10 mg/kg, every week, i.p.); Mus; RNA-Seq
Total RNA was extracted from animal tissues and cells using TRIzol reagent. For tissue samples, an appropriate amount was placed into a labeled grinding tube, 1.5 mL of TRIzol was added, and the sample was ground for 30 seconds using a tissue grinder, followed by a 5-minute incubation to ensure complete cell lysis. Alternatively, tissue samples were ground into a powder under liquid nitrogen and transferred into 1.5 mL of TRIzol in a 2 mL EP tube, followed by a 5-minute incubation. For cell samples, 1 mL of TRIzol per 1×10^6 cells was added, vortexed to mix, and incubated at room temperature for 5 minutes. The lysates were then centrifuged at 12,000 ×g for 5 minutes at 4°C. The supernatant was transferred to an EP tube containing 300 µL of chloroform/isoamyl alcohol (24:1), shaken vigorously, and centrifuged at 12,000 ×g for 8 minutes at 4°C. The aqueous phase was transferred to a new tube, mixed with isopropanol at 2/3 the volume of the supernatant, and incubated at -20°C for at least 2 hours. The mixture was centrifuged at 17,500 ×g for 25 minutes at 4°C, the supernatant discarded, and the pellet washed with 0.9 mL of 75% ethanol. After centrifuging at 17,500 ×g for 3 minutes at 4°C, the supernatant was discarded, and the pellet was air-dried for 3-5 minutes before being dissolved in 20-200 µL of DEPC-treated or RNase-free water. The library preparation process involves several key steps. First, RNA samples are quality-checked using appropriate methods based on sample type and product requirements. Next, mRNA is isolated by denaturing the RNA to disrupt secondary structures and using oligo(dT) magnetic beads to enrich for mRNA. The isolated mRNA is then fragmented using a fragmentation reagent at a specific temperature. Following fragmentation, first-strand cDNA synthesis is performed, followed by second-strand cDNA synthesis. The double-stranded cDNA is then end-repaired, an "A" base is added to the 3' ends, and adapters are ligated to the cDNA. The cDNA library is amplified by PCR, and the resulting library is quality-checked. Finally, the PCR products are denatured to single strands, circularized, and any remaining linear DNA is digested. The single-stranded circular DNA molecules are then used to form DNA nanoballs (DNBs) through rolling circle replication. These DNBs are loaded onto a high-density DNA nanochip and sequenced using combinatorial probe-anchor synthesis (cPAS) technology.