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Sample GSM1269469 Query DataSets for GSM1269469
Status Public on Dec 05, 2013
Title MCF7 total RNA
Sample type SRA
Source name MCF7 cell culture
Organism Homo sapiens
Characteristics cell line: MCF7
purification: oligo(dT) beads
sample type: input or total RNA
Treatment protocol For global mRNA for half-life measurements, MCF-7 and HEK293 cells were labeled with 700 µM 4SU for 60 min.
Growth protocol HEK293 or MCF-7 cells were grown in D-MEM high glucose with 10% (v/v) fetal bovine serum, 1% (v/v) 2 mM L-glutamine, 1% (v/v) 10,000 U/ml penicillin/10,000 µg/ml streptomycin.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using the miRNeasy kit (Qiagen). 4SU residues were biotinylated using EZ-Link biotin-HPDP (Thermo Fisher Scientific). Biotinylated 4SU-labled RNA was separated from non-labeled RNA using μMACS Streptavidin MicroBeads (Miltenyi) and 4SU-labeled RNA was eluted from μColumns by addition of 100 mM DTT. RNA was recovered from the flow-though and 4SU-labeled fractions using MinElute Spin columns (Qiagen). Input (total), flow-though (non-labeled RNA) and eluted (4SU-labled RNA) samples were used for poly(A)+ mRNA library preparation following the TruSeq RNA sample Prep v2 LS protocol (Illumina). The libraries were sequenced on an Illumina HiSeq2500 for 100 cycles (multiplexed 1× 101 + 7 index). mRNA half-lives were computed as previously described (Schwanhausser et al., 2011)
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Data processing Read demultiplexing, read filtering and adapter trimming was done using flexbar (Dodt et al, 2012).
We used TopHat2 (version 2.06) (Trapnell et al., 2009 and Kim et al. 2013) for spliced alignment of reads to the human reference genome sequence (hg18).
mRNA half-lives were determined as described in Schwanhaeusser et al. Nature 2011 from the three fractions total/4suRNA/flowthrough
Genome_build: hg18
Supplementary_files_format_and_content: text format indicating mRNA half-life in minutes for replicate 1 and 2 (rep1 and rep2) determined as described in Schwanhaeusser et al. Nature 2011
Submission date Nov 19, 2013
Last update date May 15, 2019
Contact name Markus Landthaler
Phone +49-30-9406-3026
Organization name Max-Delbrück-Center for Molecular Medicine
Department Berlin Institute for Medical Systems Biology
Street address Robert-Rössle-Straße 10
City Berlin
ZIP/Postal code 13125
Country Germany
Platform ID GPL16791
Series (1)
GSE49831 Differential Protein Occupancy Profiling of the mRNA Transcriptome
BioSample SAMN02413341
SRA SRX380130

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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