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Sample GSM665994 Query DataSets for GSM665994
Status Public on Apr 01, 2011
Title MESC_GROseq_Rep1
Sample type SRA
 
Source name embryonic stem cells
Organism Mus musculus
Characteristics strain: C57Bl/6(f) x 129/Sv(m)
cell type: V6.5 ESCs
passages: 12-14
antibody for run-on transcript enrichment: anti-Br-dUTP antibody
antibody vendor: Santa Cruz Technology, Inc.
antibody catalog #: sc-32323
Growth protocol V6.5 ESCs were grown on irradiated feeders in media that consisted of 15% FBS (Hyclone), DMEM, L-glutamine, non-essential amino acids, penicillin/streptomycin, Sodium Pyruvate (all from Invitrogen), 2-mercaptoethanol (Sigma), and LIF (Chemicon). MEFs were cultured in the same media except that 10% of FBS (Invitrogen) were used and LIF were not included. ESCs were routinely checked to confirm they remained undifferentiated by alkaline phosphatase assay and by SSEA1 immunostaining.
Extracted molecule total RNA
Extraction protocol Cells were lysed directly by adding 10 ml of lysis buffer (0.5% NP-40, 5 mM DTT, 1 mM PMSF, 10 mM Tris-Cl, pH7.4, 300 mM sucrose, 3 mM CaCl2, 2 mM MgAc2, EDTA-free protease cocktail inhibitors (Roche)) on ice for 5 min. Lysed cells were collected with scraper and spun down at 900 g for 5 min. Cell pellets in 2 ml lysis buffer were dounced 50 times with dounce homogenizer. Nuclei were collected by spinning down at 1,200 g for 5 min. Nuclei were stored in storage buffer (50 mM Tris-Cl, pH8.3, 40% glycerol, 0.1 mM EDTA, 5 mM MgAc2, 5 mM DTT, 1 mM PMSF, and EDTA-free protease cocktail inhibitors) and flash frozen in liquid nitrogen. Trypan blue staining was used to the count the number of nuclei from each preparation. the GRO-seq assay entails a short nuclear run-on using five million nuclei with 0.5 mM concentrations of Br-UTP and other NTP substrates (0.165 µM [32P]-CTP were added as a tracer) in the presence of 1% sarkosyl detergent, 5 mM MgCl2, RNase inhibitor, 1 mM DTT, 10 mM Tris-Cl, pH 8 to generate run-on transcripts about ~100 base length. After DNase I and proteinase K treatment, bulk-RNA was purified. Bulk RNA was then base hydrolyzed to about ~100 nucleotide-length with 0.2 M sodium hydroxide. Unincorporated nucleotides were removed using Biorad p30 spin column. Br-UTP-incorporated run-on transcripts were selectively enriched using anti-Br-dUTP antibody that also has high affinity to Br-UTP (Santa Cruz Technology, Inc.) three times: after hydrolysis, after 5’ adaptor ligation, and after 3’ adaptor ligation. Both ends of the RNA were repaired (using tobacco acid pyrophosphatase and T4 polynucleotide kinase) and ligated to adapters. These RNA libraries were reverse transcribed and PCR amplified non-selectively. The number of nuclei used in the reaction, run-on reaction time, base hydrolysis time, and amount of nucleotides were each titrated to ensure maximal incorporation of nucleotides with an average size of roughly 100 bases.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer
 
Description Global Run On RNA
library strategy: GRO-seq
library source: run on RNA
Data processing All fastq files have sanger-fastq format q values.
Reads were aligned to the mm9 genome assembly with eland. Reads in areas similar to rRNA sequence (as defined by RepeatMasker) were then removed.
 
Submission date Feb 03, 2011
Last update date May 15, 2019
Contact name Josh Waterfall
E-mail(s) waterfalljj@mail.nih.gov
Organization name NIH
Street address 37 Convent Dr Room 6138
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
 
Platform ID GPL9185
Series (1)
GSE27037 Regulating RNA Polymerase Pausing and Elongation in Embryonic Stem Cell Transcription
Relations
SRA SRX040536
BioSample SAMN00205387

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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