GSM9422139: Rep1_PUM2; Homo sapiens; RIP-Seq
For the reverse cross-linking reaction, lysates were mixed with 33 μL of reverse crosslinking buffer (3X PBS, 6% N-Lauroylsarcosine, 30 mM EDTA, 15 mM DTT), 10 μL of Proteinase K (Ambion), and 1 μL of RNaseOUT (Invitrogen) and incubated at 42C for 1 hour then 55C for 1 hour. TRIzol (Invitrogen) and chloroform, followed by RNeasy Plus Mini Kit (Qiagen), were used to isolate RNAs. Sequencing libraries were generated using customized primers that anneal to the universal priming sites on the oligos and attach Illumina-compatible sequencing adapters: reverse primer (AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT), forward primer (caagcagaagacggcatacgagatCGTGATgtgactggagttcagacgtgtgctcttccgatctACTGGCCGCTTCACTG, CGTGAT is a Illumina truseq index and can be variable). Amplicon sequencing was performed with a single-end run.