LO3-Env
Homo sapiens
Protocols: Two days post-transfection, cells were washed with PBS and irradiated on ice with 254 nm light (150 mJ). Cells were resuspended in lysis buffer supplemented with 1 mM PMSF and protease inhibitors (cOmplete-EDTA, Roche), before sonication on ice twice with 10-second pulses. Transcripts were fragmented for 3 minutes at 37 °C in the presence of 1 U/mL TURBO DNase and 20-1000 U/mL RNase I (Ambion). Insoluble material was pelleted by 10 minutes of centrifugation at 22000 g at 4 °C, and supernatant was added to Anti-V5 magnetic beads (Sigma, equivalent to 70 µL suspension), prewashed in lysis buffer. After binding 150 minutes on a wheel at 4 °C, RNPs were washed twice with ice-cold High-salt wash buffer and three times with ice-cold PNK buffer. Purified RNPs were radiolabelled at 5'end, resolved on Bis-Tricine 8%PAGE and transferred on nitrocellulose. Size-selected RNPs were eluted, digested with proteinase K-SDS, RNA was extracted with Phenol-Chloroform and precipitated Libraries were prepared following the iCLiP2 protocol (Buchbender et al., Methods 2020). Individual libraries were barcoded by 5'ligation of a DNA adapter to the cDNA, that includes a UMI.
ENA first public
2026-06-12
INSDC center name
University of Bergen, Michael Sars Centre
INSDC status
public
Submitter Id
E-MTAB-15845:LO3-Env
broker name
ArrayExpress
cell line
HEK293T
collection date
not collected
common name
human
development stage
foetus
geographic location (country and/or sea)
not collected
isolate
not applicable
organism part
kidney
sample name
E-MTAB-15845:LO3-Env
scientific_name
Homo sapiens