GSM7916788: SR34a-GFP iCLIP2 ABA biol rep 3; Arabidopsis thaliana; OTHER
Germinated seed tissues were UV-crosslinked (2000 mJ/cm2), frozen in liquid N2 and ground to a homogeneous powder before cell lysis. SR34a-GFP-RNA and GFP-RNA complexes were precipitated using GFP-trap agarose beads (Chromotek). After 3' end RNA linker ligation and 5'end radiolabelling, RNA-protein complexes were run on an SDS gel, transferred on a nitrocellulose membrane, and revealed by autoradiography. After a proteinase K treatment, protein-bound RNA was purified using phenol-chloroform and concentrated with the RNA Clean and Concentrator kit (Zymo Research, R1013). Library preparation was performed exactly as in Buchbender et al., 2019 (doi:10.1016/j.ymeth.2019.10.003). Purified RNAs were reverse transcribed and 5' adaptors were ligated to the subsequent cDNAs. cDNAs were then PCR-amplified. 10 nM of each library was submitted to sequencing using the NextSeq 500 system (Illumina).