title: TotalRNA_0h_n1
organism: Homo sapiens
description: Protocols: The SW1353 chondrosarcoma cell line (ATCC HTB-94) was cultured in Dulbecco's Modified Eagles Medium (DMEM, ThermoFisher Scientific, 31885023) containing 10% Foetal Bovine Serum (FBS, Sigma, F7524), 100 units/ml penicillin-streptomycin (Gibco, 15140122) and 5 µg /ml amphotericin B (Gibco, 15290018), cultured at 37 °C in a 5% CO2 environment. Serum was withdrawn when the cells had reached ~80% confluence and then 24 hours later the cells were treated with 10 ng/ml IL-1β (Sigma, I9401) for 3 and 24 hours (3x 175cm2 flasks per time point, n=3). The SW1353 chondrosarcoma cell line (ATCC HTB-94) was cultured in Dulbecco's Modified Eagles Medium (DMEM, ThermoFisher Scientific, 31885023) containing 10% Foetal Bovine Serum (FBS, Sigma, F7524), 100 units/ml penicillin-streptomycin (Gibco, 15140122) and 5 µg /ml amphotericin B (Gibco, 15290018), cultured at 37 °C in a 5% CO2 environment. Serum was withdrawn when the cells had reached ~80% confluence and then 24 hours later the cells were treated with 10 ng/ml IL-1β (Sigma, I9401) for 3 and 24 hours (3x 175cm2 flasks per time point, n=3). Ribosome profiling was carried out similar to the method described by Ingolia et al. (64), modified with the use of the ARTseq (Mammalian) Ribosome Profiling Kit (Epicentre, RPHMR12126). To harvest cells for ribosome profiling, cell culture media was removed and cells were washed with ice-cold PBS containing 0.1 mg/ml cycloheximide (Millipore UK Ltd, 239763). Cells were scraped into 800 µl mammalian lysis buffer (20% v/v 5x Mammalian Polysome Buffer (supplied in ARTseq RP Kit), 1% v/v Triton X-100 (supplied), 10 mM DTT (supplied), 10 U/µl DNase I (supplied), 0.1 mg/ml cycloheximide), then passed through a 22-25 G needle to lyse the cells completely. Cell lysate was then incubated on ice for 10 minutes and then centrifuged for 10 minutes at 20,000 xg at 4 °C. The supernatant was transferred to a pre-chilled tube and kept on ice. A 1:10 dilution of the clarified supernatant was prepared using nuclease-free water and the A260 reading of the lysate was determined using a nanodrop spectrophotometer. For each sample, the supernatant was split into 100 µl aliquots and to one aliquot, 10 µl 10% SDS was added and served as the 'Total RNA' sample. To the remaining 100 µl aliquots, 5 units of ARTseq Nuclease was added for each A260 of lysate and incubated at room temperature for 45 minutes with gentle mixing. Nuclease digest reactions were stopped by the addition of 300 U of SUPERase•In Inhibitor (ThermoFisher Scientific, AM2696). The ribosome protected fragments (RPFs) were purified according to the ARTseq Ribosome Profiling Kit (Mammalian) protocol. Briefly, the 100 µl aliquots that were nuclease digested earlier were added to Micro-Spin S-400 columns (GE Healthcare, 27-5140-01), which had been equilibrated by gravity flow with 3 ml of 1x Mammalian Polysome Buffer and centrifuged for two minutes at 600 xg. The flow-through was collected and 10 µl 10% SDS was added - this then served as the 'Ribosome Protected RNA' (RPF) sample. Circularised cDNA (5 µl) was mixed with 2 µl of the ARTseq Forward PCR Primer (supplied), 2 µl of a ScriptMiner Index PCR primer of choice (supplied index primers 1-12) and 25 µl 2x Phusion High-fidelity PCR Master Mix (New England Biolabs, M0531) in a total volume of 50 µl. Samples were then ran in following PCR program: 98 °C for 30 seconds, then 15 cycles of 94 °C for 15 seconds, 55 °C for 5 seconds and 65 °C for 10 seconds. PCR products were purified using the Agencourt AMPure XP beads (Beckman Coulter, A63880) according to the manufacturer's instructions and eluted into 25 µl nuclease-free water. To check for successful PCR amplification, 2.5 µl of the PCR amplified samples was mixed with 6x native gel loading dye and run on a Novex 8% NativeTBE gel (Thermo Fisher Scientific, EC6215BOX) at 200 V until the dye front reached the bottom of the gel, with ΦX174 RF DNA/HAE III fragments as markers (ThermoFisher Scientific, 15611-015). The gel was then stained with SYBR Gold and visualised using a dark field transilluminator. Samples that contained successfully amplified libraries (140-160 bp) were sent for QC and sequencing.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-02-01T17:01:57Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-12-04T09:42:53Z
attributes.tag: External Id
attributes.value: SAMEA5147542
attributes.tag: INSDC center name
attributes.value: University of Liverpool
attributes.tag: INSDC first public
attributes.value: 2019-02-01T17:01:57Z
attributes.tag: INSDC last update
attributes.value: 2018-12-04T09:42:53Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7466:TotalRNA_0h_n1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: SW1353
attributes.tag: cell type
attributes.value: fibroblast
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: chondrosarcoma
attributes.tag: organism part
attributes.value: bone
attributes.tag: sample name
attributes.value: E-MTAB-7466:TotalRNA_0h_n1
attributes.tag: scientific_name
attributes.value: Homo sapiens