GSM8745149: pLJM1-EGFP-2; Homo sapiens; RIP-Seq
10 million cells were collected and lysed in RIP buffer (20 mM Tris-HCl pH=7.4, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% NP-40, 100U RNase inhibitor, 1×cocktail), and lysates were incubated withGFP-beads (AlpalifeBio, ktsm1301) overnight at 4°C. Immunocomplexes were washed with washing buffer for 3 times (20 mM Tris-HCl pH=7.4, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA), treated with proteinase K, and RNA was extracted using phenol/chloroform/isoamyl alcohol (125:24:1). Clean reads were aligned to the hg38 genome using bowtie2 (v2.5.1). Both input and RIP samples were prepared for next-generation sequencing. RIP sequencing libraries were prepared using Illumina TruSeq-stranded-mRNA following manufactors' protocols.