title: Stimulated CD4 hnRNP L CLIP-seq 3
design: CD4+ T cells purified from peripheral blood mononuclear cells from three separate   anonymized donors were obtained from the University of Pennsylvania Human Immunology   Core (IRB protocol #811028). For each sample, half of the cell population was cultured in RPMI   medium containing 10% Fetal Bovine Serum at 37°C in 5% CO2 for 24 hours (resting conditions), while the other   half was stimulated with 10µg anti-CD3 and anti-CD28 antibodies for 60 hours (stimulated conditions). CLIP was   carried out according to previously published protocol {Ule, 2009 #1943;Ule, 2005 #1946}.   Briefly, resting and stimulated cells were washed twice with Hank’s Balanced Salt Solution   (HBSS), and subjected to UV-crosslinking on ice. Cells were lysed in high stringency buffer   (containing 0.1% SDS, 0.5% sodium deoxycholate, 0.5% NP40), treated with DNase, and RNase   T1, and immunoprecipitated using Protein-G dynabeads coated with anti-hnRNP L antibody   (Abcam). After stringent washing, radioactive 3’ RNA linker (RL3, GTGTCAGTCACTTCCAGCGG) was ligated to RNA cross-linked to protein, and samples resolved on 10% Bis-Tris Novex NuPAGE gels. The RNA bound to   hnRNP L was eluted from the membrane by proteinase K/7M Urea, ligated to 5’ RNA linker,   and cDNA synthesized. The library was prepared from the cDNA samples using a two-step PCR   reaction, with the second PCR step incorporating primers containing bar-codes for high   throughput sequencing.