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Sample GSM1186447

Query DataSets for GSM1186447

Status

Public on Mar 21, 2014

Title

V6.5_12.5minFP_#2

Sample type

SRA

Source name

mouse embryonic stem cells

Organism

Mus musculus

Characteristics

cell line: V6.5
treatment: treated 12.5min 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_12.5minFP_Plus.bedGraph
V6.5_12.5minFP_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