organism: Homo sapiens
description: Protocols: shControl melanoma cells cultured in 10 cm plates were washed with 4 ml cold PBS, 2ml of PBS were added and cells were irradiated at 240 nm on ice with an energy of 150 mJ/cm2 using a UVC 500 UV Crosslinker (Hoefer). Cells were collected and centrifuged for 3 min at 3000 rpm. The pellet obtained from 2.5 plates was resuspended in 500 μl of iCLIP lysis buffer (50mM Tris-HCl, pH 7,4; 100 mM NaCl; 1% Igepal CA-630; 0,1% SDS; 0,5% sodium deoxycholate). The cell lysate was sonicated using a bioruptor (Digenode), for 3 cycles of 30 seconds at level H. RNA was then partially digested by adding 10 μl of RNaseI (Ambion, AM2295) diluted 1/1000, as well as 2μl of Turbo DNase (Ambion, AM 2238). After 3 minutes of incubation at 37°C under shaking at 1100 rpm, the lysate was incubated on ice for 5 minutes, centrifuged 10 minutes at 15000 rpm and the supernatant was collected. For pre-clearing before immunoprecipitation, 500 μl lysate were diluted with 1500 μl iCLIP lysis buffer supplemented with 2 μl SUPERase-In (Ambion, AM2694) and incubated in presence of 50 μl of naked Dynabeads for 30 minutes under rotation at 4°C. For immunoprecipitation of UNR-RNA complexes, 5 μg of anti-UNR affinity purified antibody were covalently crosslinked to 40 μl of protein A magnetic Dynabeads (Invitrogen). The pre-cleared lysate was incubated with the anti-UNR beads or with beads alone as control, during 1 h under rotation at 4°C. cDNA libraries were produced as in (Konig et al 2010, Jul;17(7):909-15).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2016-11-07T17:06:43Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-09T03:49:46Z
attributes.tag: External Id
attributes.value: SAMEA3503280
attributes.tag: INSDC center name
attributes.value: Centre de Regulacio Genomica (CRG)
attributes.tag: INSDC first public
attributes.value: 2016-11-07T17:06:43Z
attributes.tag: INSDC last update
attributes.value: 2018-03-09T03:49:46Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-3818:Sample 3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: SK-MEL-103
attributes.tag: cell type
attributes.value: melanocyte
attributes.tag: common name
attributes.value: human
attributes.tag: sample name
attributes.value: E-MTAB-3818:Sample 3
attributes.tag: scientific_name
attributes.value: Homo sapiens