GSM7765985: eCLIP_MYCN_shZCCHC8_R2; Homo sapiens; OTHER
For eCLIPseq, cells were UV-crosslinked at 254 nm, lysed, sonicated in a Covaris Ultrasonicator at low settings and mixed with pre-incubated primary antibody/beads. Size-matched input samples were transfered to new tubes, IP samples were dephosphorylated and 3'RNA adapter ligated. IP and input samples were mixed with LDS buffer, loaded onto Bis-Tris gels and transfered to nitrocellulose membranes. Membrane sections were incubated with Proteinase K, input samples were dephosphorylated and 3'RNA adapter ligated. All samples were then reverse transcribed and random 5' adapters were added. A qPCR was performed to determine optimal PCR cycle number. For eCLIP, cDNA samples were amplified and gel purified. Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in paired-end mode on a NextSeq2000 platform (Illumina).