title: GSM8543099: Hepa1-6 RIP, sgUsp5, YTHDF1 _CST; Mus musculus; RIP-Seq
library_construction_protocol: Collect cell pellets, add 1 ml of IP lysis buffer (pre-supplemented with PIC and RNAse inhibitor), vortex or pipette mix thoroughly, perform on-ice lysis or rotate at 4°C for 30 minutes, centrifuge at 12, 000 x g for 10 minutes, and transfer the supernatant to a new 1.5 ml EP tube.Take 10% of the lysate volume as the input sample for RNA extraction, and assess its integrity.Take 40 μl of protein G magnetic beads, wash twice with IP buffer, add the lysateand the antibody, and incubate overnight at 4°C. Transfer the supernatant to a new 1.5 ml EP tube.Place the bead mixture on a magnetic stand, discard the supernatant once it becomes clear, add 1 ml of wash buffer, mix thoroughly, and rotate at 4°C for 10 minutes. Place the bead mixture on a magnetic stand, discard the supernatant, and repeat the washing step three times.Place the bead mixture on a magnetic stand, discard the supernatant once it becomes clear, add 1 ml of TRIzol™ Reagent (Invitrogen™, cat. no15596018), and perform RNA extraction using the phenol-chloroform method. Fragment the obtained RNA sample and prepare libraries using the EpiTM mini longRNA-seq kit:Remove ribosomal RNA using 5XRT Buffer and rRNA probes. After rRNA removal, add random primers, TSO, reverse transcriptase, and perform a 40°C reaction for 90 minutes. Inactivate the enzyme at 70°C for 15 minutes to synthesize the first-strand cDNA based on the SMART principle. Purify the cDNA using magnetic beads, add 2xpfuMax HiFi PCR ProMix and index primers for PCR amplification to enrich library fragments. Purify the DNA using magnetic beads to select library fragments.