GSM8887879: LPY-3; Rattus norvegicus; RNA-Seq
Tissue Grinding and Lysis: Tissue is ground and added to Trizol reagent. It is then incubated at room temperature for 10 minutes to allow for lysis. Phase Separation: Chloroform is added, and the mixture is vigorously shaken and centrifuged to separate the aqueous and organic phases. RNA Extraction: The aqueous phase is collected and an equal volume of phenol:chloroform is added. The mixture is centrifuged again to separate the phases. Additional Phenol:Chloroform Extraction: The aqueous phase is collected again and an equal volume of chloroform is added. The mixture is centrifuged once more to separate the phases. RNA Precipitation: The aqueous phase is collected and an equal volume of isopropanol is added. The mixture is incubated at -20°C for 1 hour and then centrifuged to precipitate RNA. Washing and Drying: The supernatant is removed, and 75% ethanol is added to wash the pellet. The mixture is centrifuged, and the supernatant is removed. Repeat Washing: Step 6 is repeated to wash the pellet again. Drying: The pellet is briefly centrifuged to remove any remaining ethanol and then dried under vacuum. RNA Resuspension: RNase-Free water is added to dissolve the RNA, and the solution is stored at -80°C. mRNA Isolation and Fragmentation: mRNA is isolated using magnetic beads with Oligo (dT) sequences. It is then fragmented and primers are added. First Strand cDNA Synthesis: The fragmented mRNA is reverse transcribed into cDNA using random primers and a reverse transcriptase enzyme. Second Strand cDNA Synthesis: The first strand cDNA is used as a template to synthesize the second strand, resulting in double-stranded cDNA. End Preparation: The ends of the double-stranded cDNA are repaired and prepared for adapter ligation. Adapter Ligation: Adapters are ligated to the ends of the cDNA molecules. Purification: The ligation products are purified using magnetic beads. PCR Amplification: The purified cDNA library is amplified using PCR with index primers to add barcodes for sample identification. Final Purification: The amplified library is purified using magnetic beads to remove any remaining impurities. Library Quality Control: The final library is assessed for size distribution and concentration using a bioanalyzer or other suitable method.