title: GSM2259091: FLAG CLIP IN Rosa-PTBP1; PTBP2 loxp/loxp; Emx1-Cre; Mus musculus; OTHER
library_construction_protocol: Mouse neocortices are triturated in ice-cold HBSS solution and UV-irradiated at 400 mJ/cm2, 4 °C in Stratalinker. Nuclear fractions are prepared as in Damianov 2016. Brain lysates were immunoprecipitated (IP) with specific antibodies. Crosslinked RNA was partially digested on the beads by micrococcal nucleases, dephosphorylated by alkaline phosphatase, ligated with a 3' linker and 5' labeled with P32-γ-ATP. The RNA-PTBP complexes were then resolved by SDS-PAGE and transferred to nitrocellulose membrane. FLAG-PTBP1 is 60 kDa and PTBP2 is 58 kDa. The nitrocellulose membrane between 80-110 kDa (i.e., the RNA-PTBP complexes) were cut and eluted the RNA with proteinase K treatment. The eluted RNA was reversed transcribed. The synthesized cDNA was circularized, linearized and amplified before sequencing on the Illumina Hi-Seq platform.
attributes.tag: GEO Accession
attributes.value: GSM2259091