title: GSM2292518: hnRNPA2/B1_eCLIP_011; Homo sapiens; RIP-Seq
library_construction_protocol: 10cm dishes of motor neuros were rinsed in PBS and UV crosslinked. Crosslinked cells were peletted and snap frozen in liquid nitrogen eCLIP libraries were prepared as previously described (Van Nostrand et al. 2016) using rabbit polyclonal antibody GTX127928 (Genetex) (10 mg per sample). Human iPSC-MNs were differentiated in culture for 28 days. Approximately 20 million cells were UV crosslinked at λ=254nm with 4000 mJ/cm2 of energy. Crosslinked cell pellets were lysed and treated with RNAse I (Ambion). Lysate was immunoprecipitated using rabbit polyclonal antibody GTX127928 (Genetex) (10 g per sample) and Dynabeads Protein G (Thermo Fisher Scientific). Immunoprecipitated samples were washed and dephosphorylated with Fast AP (Thermo Fisher Scientific). 3’ phosphates were applied using T4 PNK (NEB) and a 3’ RNA adapter was ligated using T4 RNA Ligase (NEB). Samples were run on PAGE gels and transferred to nitrocellulose membranes. A region of membrane spanning 75kDa above the molecular weight of hnRNP A2/B1 was cut and RNA was recovered using Proteinase K and phenol-chloroform. RNA was reverse transcribed using Affinityscript (Agilent Technologies) and treated with ExoSAP-IT (Affymetrix). A DNA adapter containing a 5nt randomer was then ligated to the 3’ end (T4 RNA Ligase, NEB). Samples were purified using Dynabeads MyOne Silane (Thermo Fisher Scientfic), quantified by qPCR, and then PCR amplified to the appropriate number of cycles. Amplified libraries were size selected with an agarose gel, quantified using the Agilent Tapestation, and sequenced on the Illumina HiSeq 2500 platform using paired end 50bp reads.