title: GSM8596695: TFEB41; Mus musculus; RNA-Seq
library_construction_protocol: Appropriate amount of tissue sample was taken into the corresponding number of grinding and crushing pipe. Add 1.5mL TRIzol lysate, put it into a tissue grinder, grind it for 30s, take it out and leave it for 5min to fully lysate the tissue cells. The liquid nitrogen was ground and crushed, and 1.5mL TRIzol lysate was divided into 2mL EP tubes. An appropriate amount of tissue sample was taken and ground into powder under the protection of liquid nitrogen, and then transferred into the lysate. Lay flat for 5min to fully lyse the tissue cells. Appropriate amount of TRIzol lysate was added into the cell sample tube (the reference dosage was 1 mL of TRIzol for every 1×106 cells), and the cells were placed at room temperature for 5min after swirling and mixing, so as to fully lysate the cells. The ground and broken tissue samples were centrifuged (cell samples did not need to be centrifuged) at 4℃ for 5min at 12000 ×g. The supernatant was transferred into an EP tube filled with 300µL chloroform/isoamyl alcohol (24:1), reversed and violently shook and mixed; Centrifuge at 4℃ at 12000×g for 8min. If the middle layer is thick and the water is turbidity, the same supernatant volume of chloroform/isoamyl alcohol (24:1) can be extracted again. Absorb the supernatant into a new 1.5mL centrifuge tube, add isopropyl alcohol (isopropyl alcohol) containing 2/3 of the supernatant liquid (2µL 5mg/mL glycogen can be added during precipitation), invert and mix gently, and place in the refrigerator at -20℃ for more than 2h. Centrifuge at 4℃ at 17500×g for 25min, discard the supernatant, wash with 0.9mL 75% ethanol, suspend precipitation upside down, centrifuge at 4℃ at 17500×g for 3min(depending on the precipitation, it can be washed again with 75% ethanol, centrifuge at 4℃ at 17500×g for 3min). Discard the supernatant, remove the residual liquid after a short centrifugation, and let dry for 3-5min. Dissolve precipitation with 20-200μL DEPC water or RNase-free water. Sample testing According to the sample and product requirements to select the appropriate test method for quality inspection. A certain amount of RNA samples were isolated, the secondary structure was opened by thermal denaturation, and the mRNA was enriched by oligo(dT) magnetic beads. mRNA interruption The mRNA obtained in the previous step is fragmented by adding an interruption reagent to the mRNA obtained in the previous step and reacting at a certain temperature for a certain time. A chain synthesis reaction system was prepared for cDNA synthesis, and the reaction program was set to synthesize a chain cDNA. The two-strand synthesis reaction system was prepared and the reaction program was set up to synthesize two-strand cDNA. The reaction system was prepared by terminal repair, adding "A" and joint connection, and the reaction program was set to repair the end of double-stranded cDNA and add A base to the 3 'end. The joint joint reaction system was prepared, and the reaction program was set to connect the joint with cDNA. PCR The PCR reaction system was prepared, and the reaction program was set to amplify the product. Library testing According to the product requirements to select the corresponding detection method to the library quality inspection. After the PCR product was denaturated to single strand, the cyclization reaction system was prepared, and the reaction program was set to obtain the single strand ring product, and the linear DNA molecules that were not cycled were digested. Single-stranded circular DNA molecules are replicated by rolling rings to form a DNA nanosphere (DNB) containing multiple copies. The DNBs obtained were added to the mesh holes on the chip using high-density DNA nanochip technology, and sequenced by combined probe anchoring polymerization (cPAS).