title: ARPP21 iCLIP replicate 4
organism: Homo sapiens
description: Protocols: Flp-In-TREx-293 cells from ThermoScientific were used to generate cell lines expressing murine full-length ARPP21 upon doxycycline addition (iARPP21 cells). ARPP21 was cloned in the pENTR4 vector and transferred by Gateway cloning into the pFRT-FLAG-HA-DEST vector to provide N-terminal FLAG and HA tags and flanking FRT sites necessary for Flp recombination. Cloning and cell line generation was accomplished following Spitzer et al., 2013 (http://dx.doi.org/10.1016/B978-0-12-418687-3.00008-2). iARPP21 cells were seeded in DMEM with 10% FBS and 1 µg/ml Doxycycline. 48h after seeding, the cells were subjected to UV-crosslinking and harvested. iCLIP was performed with the mouse monoclonal HA antibody #26183, Thermo Scientific, following the improved protocol fromHuppertz et al, 2014 (http://dx.doi.org/10.1016/j.ymeth.2013.10.011). A detailed protocol with all modifications of the initial protocol can be found under Supplementary Methods. The retrieval of protein-bound RNAs and construction of Illumina compatible libraries was done as described in Huppertz et al, 2014 (http://dx.doi.org/10.1016/j.ymeth.2013.10.011).
attributes.tag: ENA first public
attributes.value: 2018-02-23
attributes.tag: ENA last update
attributes.value: 2017-07-21
attributes.tag: External Id
attributes.value: SAMEA104169553
attributes.tag: INSDC center alias
attributes.value: Bioinformatics Group, Department of Computer Science, University of Freiburg, Freiburg, Germany
attributes.tag: INSDC center name
attributes.value: Bioinformatics Group, Department of Computer Science, University of Freiburg, Freiburg, Germany
attributes.tag: INSDC first public
attributes.value: 2018-02-23T17:01:57Z
attributes.tag: INSDC last update
attributes.value: 2017-07-21T16:02:01Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5911:ARPP21 iCLIP replicate 4
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HEK293T
attributes.tag: common name
attributes.value: human
attributes.tag: genotype
attributes.value: doxycycline-inducible expression of HA-tagged ARPP21
attributes.tag: replicate
attributes.value: 4
attributes.tag: sample name
attributes.value: E-MTAB-5911:ARPP21 iCLIP replicate 4