AA0779E_eCLIP
eCLIP was performed using the eCLIP Library Prep Kit (Eclipse Bioinnovations). 1  107 AA0779E cells were UV-crosslinked and lysed. RNA was fragmented, 2% of each sample was stored for preparation of a size-matched input library. The remaining lysates were IPd using an hnRNP F antibody. Bound RNA fragments in the IPs were dephosphorylated and 3-end ligated to an RNA adapter. Protein-RNA complexes from IPs and inputs were run on an SDS-PAGE gel and transferred to a nitrocellulose membrane. Membrane regions spanning from the hnRNP F band (~46 kDa) to the first non-specific band noted in the IgG control (~82 kDa) were excised and RNA released from the complexes with proteinase K. Input samples were dephosphorylated and 3-end ligated to an RNA adapter. Both IP and input samples were reverse transcribed with AffinityScript (Agilent). cDNAs were 5-end ligated to a DNA adaptor. cDNA yields were quantified by qPCR and 100-500 fmol of libraries generated with Q5 PCR mix (NEB). Libraries were sequenced on an Illumina HiSeq2500 using dual barcoded multiplexing and a paired-end 75 bp length.
AA0779E_eCLIP