title: iCLIP of AtGRP8-GFP plants harvested from 16 days old seedlings after 24 hours of constant light (LL)
design: Seedlings on plates were subjected to irradiation with 254 nm UV light at a dose of 500 mJ/cm2 in a UVP CL-1000 UV crosslinker (on ice). Cell lysates from aerial tissue were prepared, precleared with sepharose beads and subjected to immunoprecipitation with GFP Trap beads (Chromotek) or mock precipitation with RFP Trap beads (Chromotek). On the beads, the precipitate was treated with DNase (Thermo Scientific). Subsequently, the RNAs were dephosphorylated and the L3 linker was ligated to the 3ends using RNA ligase (NEB). 5 termini were labeled using [-32P] ATP and polynucleotide kinase and the covalently linked RNAprotein complexes were separated on a 4-12 % NuPAGE Bis-Tris gel (Thermo Scientific), and electroblotted onto a nitrocellulose membrane. Upon autoradiography, the regions above the fusion protein were cut out and subjected to proteinase K treatment, leaving a polypeptide at the interaction site. Subsequently, RNA was isolated from the membrane using TriReagent. RNA was reverse transcribed using primers containing a cleavable adapter region and individual barcode sequences. After NaOH treatment, the cDNA was purified on a 6% urea-polyacrylamide gel and fragments in the size range of approx. 7085 nt (high, H), 85120 nt (medium, M), and 120200 nt (low, L), respectively, were eluted from the gel. The cDNAs were then circularized using CircLigase II (Epicentre) and an oligonucleotide (Cut-oligo) was annealed to generate a BamHI restriction site. Relineariztion via BamHI digestion results in adapters at both ends of the cDNA which then were PCR-amplified. After PCR optimization the three size fractions (H, M, L) are pooled with a ratio of 1:1:1, concentrations were assessed with Qubit dsDNA HS Assay Kit (Thermo Scientific).10 nM of the libraries were submitted to high throughput sequencing after multiplexing of multiple samples. Sequencing was carried out using an Illumina HiSeq2500 (Eurofins) with 50 nt single end or 100 nt single end reads 1
library_name: AtGRP8_LL24_1