GSM8015025
Approximately 20 million cells were UV crosslinked at 400 mJoules/cm2 with 254 nm radiation and stored at -80C until use. Cell pellets (20 million cells per pellet) were lysed using 1mL of eCLIP lysis mix with a modified composition of 6mL of Proteinase Inhibitor Cocktail and 20mL of Murine RNase Inhibitor. Ligations were performed on-bead (to allow washing away unincorporated adapter) in high concentration of PEG8000, which improves ligation efficiency to >90%. Samples were then run on standard protein gels and transferred to nitrocellulose membranes, and a region 75 kDa (∼220 nt of RNA) above the protein size was isolated and proteinase K (NEB) treated to isolate RNA. RNA was reverse transcribed with AffinityScript (Agilent), and treated with ExoSAP-IT (Affymetrix) to remove excess oligonucleotides. A second DNA adapter (containing a random-mer of 5 (N5) or 10 (N10) random bases at the 5′ end) was then ligated to the cDNA fragment 3′ end (T4 RNA Ligase, NEB), performed with high concentration of PEG8000 (to improve ligation efficiency) and DMSO (to decrease inhibition of ligation due to secondary structure). After cleanup (Dynabeads MyOne Silane, ThermoFisher), an aliquot of each sample was first subjected to qPCR (to identify the proper number of PCR cycles), and then the remainder was PCR amplified (Q5, NEB) and size selected via agarose gel electrophoresis.