title: SSA_BrU
organism: Homo sapiens
description: Protocols: Cells were harvested by scraping. HeLa cells (800000 in a 6 cm plate) were co-treated with 2 mM BrU (5-Bromouridine, Sigma-Aldrich) and 10 µM Sudemycin C1, 2 µM Sudemycin K or 5 nM Spliceostatin A for 3 hours. Total RNA was extracted using the Maxwell RNAeasy kit (Promega). Total RNA was kept for standard RNA sequencing. For BrU-RNA sequencing, 10 µg of total RNA were immunoprecipitated with a mouse anti-BrU antibody (B2531, Sigma). Briefly, 2 µl of antibody were incubated with 20 µl Protein G Dynabeads (Invitrogen) per sample for 1 h at 4°C in 1 ml RSB-100 buffer (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2.5 mM MgCl2 and 0.4% (v/v) Triton X-100). After three washes of the beads with 1 ml of RSB-100 buffer, beads were resuspended in 150 µl RSB-100 with 40 U RNasin (Promega). RNA was incubated for 1 min at 80°C, added to the beads and incubated for 1 h at 4°C. After three washes with 1 ml of RSB-100 buffer, the RNA bound to the beads was eluted by direct addition of 300 µl RLT buffer (Qiagen RNeasy mini kit). The mix was then heated at 80°C for 10' and after centrifugation the BrU-labeled RNA present in the supernatant was purified using the RNeasy mini kit (Qiagen). TruSeq Stranded mRNA Library Preparation Kit (Illumina)
attributes.tag: ENA first public
attributes.value: 2017-10-29
attributes.tag: ENA last update
attributes.value: 2017-10-19
attributes.tag: External Id
attributes.value: SAMEA104356092
attributes.tag: INSDC center alias
attributes.value: CRG
attributes.tag: INSDC center name
attributes.value: Centre for Genomic Regulation, Barcelona
attributes.tag: INSDC first public
attributes.value: 2017-10-29T17:01:48Z
attributes.tag: INSDC last update
attributes.value: 2017-10-19T16:30:38Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-6060:SSA_BrU
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HeLa
attributes.tag: cell type
attributes.value: epithelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: disease
attributes.value: adenocarcinoma
attributes.tag: organism part
attributes.value: uterine cervix
attributes.tag: sample name
attributes.value: E-MTAB-6060:SSA_BrU
attributes.tag: sampling time point
attributes.value: 3
attributes.tag: sex
attributes.value: female