GSM4513674
GSM4513674: HEK293-Trex cells, NYN2_IP; Homo sapiens; RIP-Seq
Lysates were clarified from sonicated UV-crosslinked cells and MARF1-RNA complexes were isolated with FLAG antibody. Crosslink immunoprecipitation experiments were conducted using a modified single-end (se)CLIP approach  (Van Nostrand et al., 2017).  All experiments were conducted using HEK293 cells (negative control), or HEK293 cells that express FLAG-tagged MARF-NYN.  Briefly, FLAG-tagged MARF1-NYN expression was induced in a 15 cm dish of HEK293 cells with 100ng/mL of doxycycline for 12 hours.  Following induction, 2x10e7 cells were lysed in iCLIP lysis buffer and sonicated (BioRuptor).  Lysates were treated with RNAseI (Thermo) to shear RNA, after which FLAG-tagged MARF1-NYN-RNA complexes were immunoprecipitated with FLAG antibody.  Stringent washes were performed, during wish RNA was dephosphorylated with FastAP (Thermo) and T4 PNK (NEB).  Following dephosphorylation, a 3' RNA adaptor was ligated onto the RNA with T4 RNA ligase (NEB).  Protein-RNA complexes were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and RNA was isolated from the membrane region corresponding to the migration of MARF1 and 75kDa above.  Isolated RNA was subsequently reverse transcribed with AffinityScript (Agilent) and the 3' DNA adaptor was ligated onto the cDNA with T4 RNA ligase (NEB).  Libraries were then amplified with Q5 PCR mix (NEB).
GEO
GSM4513674
GEO Accession
GSM4513674