title: Resting JSL1 hnRNP L CLIP-seq 1
design: JSL1 T cells were cultured in RPMI medium containing 10% fetal bovine serum at 37 degrees Celcius in 5% CO2 for 24 hours (resting conditions).  CLIP-seq libraries were prepared as follows: first, cells were washed twice with Hank's Balanced Salt Solution (HBSS), then cells were subjected to crosslinking by exposure to ultraviolet light of 254nm wavelength on ice.  Cells were lysed in buffer containing 0.1% SDS, 0.5% sodium deoxyholate, and 0.5% NP40.  Lysed cells were then treated with DNase and RNase T1, then subjected to immunoprecipitation using Protein-G dynabeads coated with anti-hnRNP L antibody (4D11) (Abcam, Cambridge, UK).  Immunoprecipitates were stringently washed, then radioactive 3' RNA linker (RL3, GTGTCAGTCACTTCCAGCGG) was ligated to the crosslinked RNAs.  Radiolabeled RNA-hnRNP L complexes were resolved on 10% bis-tris Novex NuPAGE (Life Technologies, Carlsbad, CA).  hnRNP L-bound RNA was eluted from the membrane with proteinase K and 7M urea, ligated to 5' RNA linker, and reverse transcribed to cDNAs.  cLIP-seq library was prepared from the cDNA samples using a two-step PCR reaction, with the second step incorporating primers containing bar codes.