GSM8389478: BMDM, 24h, RRP1,RIP,replicate3; Mus musculus; RIP-Seq
Cells were harvested using RIP lysis buffer. A primary antibody that recognized RRP1 or control IgG was added to the supernatants overnight at 4°C. Protein A/G Magnetic Beads were then added and gently mixed for 1 h at 4°C. The beads were washed with NT2 buffer 5 times before digestion with Proteinase K in buffer for 30 min at 55°C to remove proteins. The RNA was isolated using TRIzol LS and further assessed by high-throughput sequencing. The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads (foreukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp). Then the first strand of cDNA is synthesized by using randomhexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. Endreparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCRamplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library.