GSM7978762: ST-3; Mus musculus; RIP-Seq
RNA immunoprecipitation (RIP) was performed using the EZ-Magna RIP Kit (Millipore, Burlington, MA, United States) in accordance with the manufacturer's protocol. In brief, lysis buffer was used to dissolve RAW264.7 cells to prepare a lysate. A monoclonal antibody against NLRP3 was then added to the lysate and then co-precipitated with protein A/G magnetic beads overnight. The next day, protein was removed with protease K and total RNA was extracted using a mixture of phenol, chloroform, and isoamyl alcohol at 125:24:1 and pH 4.3. The library construction was completed according to the instructions of the reagent kit. Include, mRNA in total RNA enrichment using NEB Next ® Poly (A) mRNA Magnetic Isolation Module reagent or RiboZero Magnetic Gold Kit was used to remove rRNA. Afterwards, the library was constructed using KAPA Stranded RNA Seq Library Prep Kit (Illumina). Briefly, the RNA products obtained from mRNA enrichment (or rRNA removal) treatment are reverse transcribed to generate first stranded cDNA, which is then added to dUTP to synthesize double stranded cDNA. Each polymerase acts to repair the end of the double stranded cDNA and adds A, followed by ligases connecting to Illumina specific ligands, and finally PCR amplification and purification to obtain the final RNA sequencing library.