title: Illumina HiSeq 1500 sequencing; hnRNP A1 iCLIP
design: hnRNP A1 iCLIP
library_construction_protocol: T7-tagged hnRNP A1-expressing HeLa Cells were seeded in 10 cm dishes and induced with 1 µg/ml doxycycline (Sigma-Aldrich, www.sigmaaldrich.com) for 48 hours to 90% confluenceand before UV crosslinking. iCLIP was performed as described (iCLIP: protein-RNA interactions at nucleotide resolution by Huppertz et al. 2014, Methods, 65(3):274-87) with three biological replicates. Briefly, HeLa cells with stable inducible expression of T7-tagged hnRNP A1 were cultured in RPMI 1640 with L-Glutamine (Lonza, www.lonza.com) supplemented with streptomycin, penicillin and glutamine. Cells were seeded in 10 cm dishes and induced with 1 µg/ml doxycycline (Sigma-Aldrich, www.sigmaaldrich.com) for 48 hours before UV crosslinking. RNase Cocktail™ Enzyme Mix (Life Technologies, www.lifetechnologies.com), a mixture of RNaseA and RNaseT1 was used for RNA digestion. It was diluted 1:10, 1:200 and 1:2000. Protein G Dynabeads (Life Technologies) were coupled to T7-antibody (69522-4, Merck Millipore, www.merckmillipore.com/) and RNA-protein complexes were isolated and purified. cDNA was synthesized with Superscript II Reverse Transcriptase (Life Technologies). After PCR amplification, DNA fragments below 100 bp were removed by purification with Agencourt AMPure XP beads (Beckman Coulter, https://www.beckmancoulter.com).