GSM8503105: p_LOVO_H1047R_1; Homo sapiens; RNA-Seq
Sample Disruption and Lysis 1. Place an appropriate amount of tissue sample into a correspondingly numbered grinding tube (tissue ≤30mg, cells ≤1 x 10^7). 2. Add 600 µL Buffer RLT Plus, homogenize with a homogenizer, and let it sit at room temperature for 5 minutes. 3. Centrifuge at 12,000 x g for 3 minutes at room temperature; transfer the entire supernatant to an AllPrep DNA spin column. 4. Centrifuge at 10,000 x g for 30 seconds at room temperature; collect the fluid in the collection tube for RNA purification.  RNA Purification 1. Add an equal volume of 70% ethanol to the collection tube and mix well by pipetting. 2. Transfer 700 µL of the mixture to an RNeasy spin column set up with a collection tube, centrifuge at 10,000 x g for 30 seconds at room temperature; repeat this step until all the mixture has passed through the RNeasy spin column. 3. Add 700 µL Buffer RW1 to the RNeasy spin column, centrifuge at 10,000 x g for 30 seconds at room temperature, discard the waste fluid. 4. Add 500 µL Buffer RPE to the RNeasy spin column, centrifuge at 10,000 x g for 30 seconds at room temperature, discard the waste fluid. 5. Add 500 µL Buffer RPE to the RNeasy spin column, centrifuge at 10,000 x g for 30 seconds at room temperature, discard the waste fluid. 6. Centrifuge at 16,000 x g for 2 minutes at room temperature, transfer the RNeasy spin column to a 1.5 mL centrifuge tube. 7.  Add about 35 µL RNase-free water to the RNeasy spin column, let it sit at room temperature for 1 minute. 8. Centrifuge at 16,000 x g for 2 minutes at room temperature to collect RNA. 1. Sample Testing Select the appropriate testing method for quality control based on the sample and product requirements. 2. mRNA Isolation Take a certain amount of RNA sample, denature it at suitable temperature to open its secondary structure, and enrich mRNA using oligo(dT) magnetic beads. 3. mRNA Fragmentation Add fragmentation reagent to the obtained mRNA from the previous step, react at a suitable temperature for a set period, and fragment the mRNA. 4. cDNA Synthesis Prepare a reaction system for single-strand cDNA synthesis and set the reaction program; prepare a reaction system for double-strand cDNA synthesis and set the reaction program. 5. End Repair, 'A' Tailing, and Adaptor Ligation Prepare a reaction system and set the reaction program to repair the ends of the double-stranded cDNA and add an 'A' base to the 3' end; prepare an adaptor ligation reaction system and set the reaction program to ligate the adaptor to the cDNA. 6. PCR Prepare a PCR reaction system and set the reaction program to amplify the product. 7. Library Testing Select the appropriate testing method for quality control of the library based on product requirements. 8. Circularization Denature the PCR products into single strands, then prepare a circularization reaction system and set the reaction program to obtain single-stranded circular products and digest un-circularized linear DNA molecules. 9.Sequencing Single-stranded circular DNA molecules undergo rolling circle replication to form a DNA nano-ball (DNB) containing multiple copies. The obtained DNBs are applied to a high-density DNA nanoarray technology, placed into the mesh-like pores on the chip, and sequenced using the combined probe anchor synthesis technology (cPAS).