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Sample GSM1186451 Query DataSets for GSM1186451
Status Public on Mar 21, 2014
Title V6.5_50minFP_#2
Sample type SRA
 
Source name mouse embryonic stem cells
Organism Mus musculus
Characteristics cell line: V6.5
treatment: treated 50min with 300nM flavopiridol
sample type: nascent RNA
Treatment protocol Cells were treated with 300nM flavopiridol for 2, 5, 12.5, 25 and 50 min, or with 500nM triptolide for 12.5, 25, and 50 min, or with 0.0125% DMSO for 50min.
Growth protocol Cell culturing of the V6.5 mES cell line was done as in (Monkhorst et al., 2008), and drug treatment was performed on pre-plated mES cells, grown for one passage before isolation of nuclei to remove irradiated MEF-feeder cells.
Extracted molecule total RNA
Extraction protocol Nuclei isolation are performed using standard protocols, in brief, after rinsing the 15 cm2 plates with drug-treated cells, 15 ml cell lysis buffer (10 mM Tris-Cl, pH 7.5, 300 mM Sucrose, 3mM CaCl2, 2 mM MgAc2, 0.5% NP-40, 5 mM DTT, 1 mM PMSF, protease inhibitors) was added to the plates and the cells were scraped off, and spun down at 4°C and 185 xg for 5 min in a GS-6R Beckman swing-bucket centrifuge, after which supernatant was discarded. 5 ml fresh cell lysis buffer was added and cells were dounced 50 times in a 5 ml douncer on ice and spun down at 328 xg for 5 min, after which supernatant was discarded and nuclei were taken up in ~250 μl of glycerol storage buffer (50 mM Tris-Cl, pH 8.3, 40% glycerol, 0.1 mM EDTA, 5 mM MgAc2, 5 mM DTT, 1 mM PMSF, protease inhibitors) and snap frozen.
For each nuclear run-on (NRO), 1e7 nuclei were used. An NRO master mix was added containing nucleotides ATP, GTP, CTP and nucleotide analogue bromo-UTP, as well as sarkosyl and 300 mM KCl to remove all impediments that pause Pol II and prevent NRO and to prevent re-initiation of Pol II. The NRO was performed at 37°C for 5 min, which results in ~100 nt addition to the nascent RNA. Immediately after the NRO, a population of ~100 different in-vitro transcribed Arabidopsis thaliana spike-in RNAs with and without Br-UTP was added to the nascent RNA as a way to assess experimental differences between each library, and to use as a normalization tool between timepoints within each timecourse. The nascent RNA was fragmented to ~150 nts and NRO BrU-RNA was isolated 3 consecutive times with BrdU-antibody beads (sc-32323, Santa Cruz), with enzymatic TAP and PNK treatments to remove the cap and 3’-phosphate and to add a 5’-phosphate, as well as Illumina adaptor ligations between the BrU-RNA isolation steps. The three consecutive isolation steps lead to an approximate 500.000x enrichment of BrU-RNA over background RNA. BrU-RNA was reverse transcribed, amplified, barcoded and sent for sequencing. Each dataset was done in replicate.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 1000
 
Description GRO-seq
V6.5_50minFP_Plus.bedGraph
V6.5_50minFP_Minus.bedGraph
Data processing All the GRO-seq libraries were sequenced in 50 nt runs on the Illumina HiSeq and split by barcode. Sequences that contained the Illumina adaptor were clipped with the cutadapt tool (https://code.google.com/p/cutadapt/) and remaining sequences were trimmed down to 32-mers, aligned to the ribosomal genome to remove rRNAs, and then aligned uniquely to the mm9 reference genome, with up to 2 mismatches with bowtie (http://bowtie-bio.sourceforge.net/index.shtml). Replicates were highly correlated and were pooled for further analysis, with exception of one of the 25 min Trp-treated #1 replicate, which suffered from extensive RNA degradation during the library preparation, and was subsequently discarded. Spike-in RNA controls were aligned similarly to a spike-in reference genome.
Sequences that contained the Illumina adaptor were clipped with the cutadapt tool (https://code.google.com/p/cutadapt/) and remaining sequences were trimmed down to 32-mers,
32-Mers were aligned to the ribosomal genome to remove rRNAs, and then aligned uniquely to the mm9 reference genome, with up to 2 mismatches with bowtie (http://bowtie-bio.sourceforge.net/index.shtml).
Replicates were highly correlated and were pooled for further analysis, with exception of one of the 25 min Trp-treated #1 replicate, which suffered from extensive RNA degradation during the library preparation, and was subsequently discarded. Spike-in RNA controls were aligned similarly to a spike-in reference genome.
Instead of using total read counts to normalize between treatments within either the FP or Trp time course, we chose to normalize with the spike-in RNA read counts instead.
Genome_build: mm9
Supplementary_files_format_and_content: bedGraph, nascent RNA read density of combined replicates, normalized to spike-in controls for each time course. Split by Plus and Minus strand
 
Submission date Jul 15, 2013
Last update date May 15, 2019
Contact name Iris Helene Jonkers
Organization name Cornell University
Department Molecular Biology & Genetics
Lab Lis Lab
Street address Biotechnology Building 417
City Ithaca
State/province NY
ZIP/Postal code 14853
Country USA
 
Platform ID GPL15103
Series (1)
GSE48895 Genome-wide dynamics of Pol II elongation and its interplay with promoter proximal pausing, chromatin, and exons
Relations
BioSample SAMN02251428
SRA SRX323000

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

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