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Sample GSM2835841 Query DataSets for GSM2835841
Status Public on Mar 06, 2018
Title MEF_GROSeq_KO2_19966
Sample type SRA
Source name primary mouse embryonic fibroblasts
Organism Mus musculus
Characteristics tissue: primary mouse embryonic fibroblasts
genotype: Supt5hFl/- Rosa26ERT2-cre/+
Treatment protocol Primary MEFs were treated with 2 uM 4-HT for 72h and then harvested for all downstream applications.
Growth protocol Primary MEFs were prepared from d13.5 Supt5hFl/+Rosa26ERT2-cre/+ and Rosa26ERT2-cre/+ embryos and cultured in complete DMEM medium to passage 3.
Extracted molecule total RNA
Extraction protocol Following cell membrane lysis with hypotonic buffer, nuclei were isolated via sucrose gradient density centrifugation. GROseq was performed as described (Core & Lis, 2008). Briefly, 10 million nuclei were used per reaction, and the run-on was performed for 5 minutes with NTPs with a limiting concentration of P32-dCTP and dBrdUTP followed by extraction of RNA with Trizol reagent (Thermo Fisher, 15596026). An updated protocol was kindly provided by Dr. John Lis (Cornell University).
After base hydrolysis and DNA end repair, Illumina compatible 3' RNA adapters were ligated to total RNA. After BrdU- incorporated RNA IP the 5' cap was removed and the 5' DNA end was repaired. Next, the 5' DNA Illumina comaptible adapters were ligated, and the library was reverse transcribed and amplified.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
Description in vitro transcribed RNA
Data processing Library strategy: GRO-Seq
Adapters were trimmed from the 3' ends of the reads with cutadapt (v 1.10), and a 5nt random linker was removed from the 3' end using fastx_trimmer.
Trimmed reads larger than 10nt were aligned to the genome using bowtie v1.0.0 (-v 2 --best --strata -- tryhard -m 1).
genome build: NCBI mm9
processed data files format and content: bigWig of rpm-normalized read densities
Submission date Oct 30, 2017
Last update date May 15, 2019
Contact name Tobias Neumann
Organization name IMP
Street address Campus-Vienna-Biocenter 1
City Vienna
ZIP/Postal code 1030
Country Austria
Platform ID GPL13112
Series (1)
GSE106313 Regulation of RNA polymerase II processivity by Spt5 is restricted to a narrow window in elongation
BioSample SAMN07946539
SRA SRX3342585

Supplementary file Size Download File type/resource 63.4 Mb (ftp)(http) BW 62.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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