title: GSM8122110: RS2Z35 iCLIP2 Replicate 3; Solanum lycopersicum; OTHER
library_construction_protocol: Cells were lysed and RNA-Protein-complexes were immunoprecipitated with GFP-Trap magnetic agarose beads (GFP Nanobody-coupled beads) and subjected to on-bead RnaseI treatment. After stringent washing, RNA was dephosphorylated and ligated to a 3' linker. Protein-RNA complexes were resolved by denaturing gel electrophoresis and transfered to nitrocellulose membrane. A membrane region above the expected size of the protein was cut and RNA was released by Proteinase K treatment. RNA was reverse transcribed using reverse primer and a second adapter containing a barcode was ligated to the cDNAs. The cDNA was pre-amplified for 6 cycles using short Solexa P3 and P7 primers. After a size selection on beads a second PCR amplification was performed with the normal P3 and P7 primers to generate the final iCLIP2 libraries.