title: FXR1-WT
organism: Homo sapiens
description: Protocols: HeLa were grown in 10cm dishes to ~70% confluency and then transfected with either mGFP-FXR1-WT or mGFP-FXR1-V361Pmut for 20 hours. Cells were irradiated once with 150 mJ/cm2 in a Spectroline UV Crosslinker at 254 nm. Cell pellets were snap-frozen 0.1 Units of RNaseI and 4 Units Turbo DNase were added per 1 ml of cell lysate at 1mg/ml protein concentration for RNA fragmentation. Antibodies against GFP (ab290) coupled to magnetic Protein G beads were used to isolate Protein-RNA complexes, and RNA was ligated to a pre-adenylated infra-red labelled IRL3 adaptor. The complexes were then size-separated by SDS-PAGE, blotted onto nitrocellulose and visualised by iBright scanning. RNA was released from membrane by proteinase K digestion and recovered by precipitation. cDNA was synthesized with Superscript IV Reverse Transcriptase (Life Technologies) and AMPure XP beads purification (Beckman- Coulter, USA), then circularised using Circligase II (Epicentre) followed by AMPure XP beads purification. After PCR amplification, libraries were size-selected with Ampure beads and quality controlled for sequencing.
attributes.tag: disease
attributes.value: adenocarcinoma
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: organism
attributes.value: Homo sapiens
attributes.tag: collection date
attributes.value: not collected
attributes.tag: sex
attributes.value: female
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: common name
attributes.value: human
attributes.tag: organism part
attributes.value: cervix
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: endogenous FX1 knock down and transfection with mGFP-FXR1-WT
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: cell_line
attributes.value: HeLa
attributes.tag: genetic modification
attributes.value: WT
attributes.tag: cell_type
attributes.value: epithelial cell
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2023-11-17