GSM9151004: Wild-type mouse liver on HFD, replicate 3; Mus musculus; RNA-Seq
The process begins with tissue lysis, where the liver sample is ground using a grinding machine, then transferred to a tube containing 1.5 mL of TRIzol lysis buffer. The mixture is ground for 30 seconds and left to stand for 5 minutes to ensure complete tissue cell lysis. Following lysis, the sample is centrifuged at 12000×g for 5 minutes at 4℃ to pellet cellular debris. The resulting supernatant is then transferred to a new tube, mixed thoroughly with 300 μL of Chloroform/isoamyl alcohol (24:1), and centrifuged again at 12000×g for 8 minutes at 4℃ to separate the phases. The aqueous supernatant, containing the target molecules, is carefully transferred to a fresh tube. To this, 2/3 volume of Isopropyl alcohol is added (with optional glycogen for low-input samples), and the mixture is gently inverted and incubated at -20℃ for over 2 hours to precipitate the molecules. After precipitation, the sample is centrifuged at 17500×g for 25 minutes at 4℃ to form a pellet. The supernatant is discarded, and the pellet is washed with 0.9 mL of 75% ethanol by inversion. A final centrifugation at 17500×g for 3 minutes at 4℃is performed to remove the remaining wash buffer. The final pellet is air-dried for 3-5 minutes to remove residual ethanol and then dissolved in 20-200 μL of DEPC-H2O or RNase-free water for downstream analysis. The protocol begins with a quality control check of the initial RNA sample to ensure it meets product specifications. Next, mRNA is isolated by denaturing the RNA sample at an appropriate temperature to disrupt its secondary structure, followed by enrichment using oligo(dT) magnetic beads. The purified mRNA is then fragmented into smaller pieces using a fragmentation reagent at a suitable temperature. This is followed by cDNA synthesis, where a reaction system is prepared and programmed to synthesize the first cDNA strand, and then a separate reaction system is used to synthesize the second strand. The double-stranded cDNA then undergoes end repair to create blunt ends, followed by the addition of an "A" base to the 3' end. Adapter ligation is performed by preparing a reaction mixture and program to attach adapters to the cDNA fragments. The resulting product is then amplified using PCR with a prepared reaction system and program. The amplified library is then subjected to a quality control check. In the cyclization step, the PCR product is denatured into single strands, and a cyclization reaction is performed to create single-stranded circular DNA molecules, with unligated linear DNA molecules digested away. Finally, for sequencing, the single-stranded circular DNA molecules undergo rolling circle replication to form DNA nanoballs (DNBs). These DNBs are loaded onto a high-density DNA nan chip and sequenced using combinatorial probe-anchor synthesis (cPAS) technology.