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Sample GSM3045646

Query DataSets for GSM3045646

Status

Public on Mar 17, 2018

Title

4sURNA_60_R1

Sample type

SRA

Source name

mESC_4sU_60

Organism

Mus musculus

Characteristics

rna: Labeled
labeling time (min): 60
biological replicate: 1

Treatment protocol

4sU (Sigma, T4509) was added to the growth medium (final concentration of 200 µM) and the cells were incubated at 37 ºC for 15, 30 or 60 minutes. Plates were washed once with 1X PBS and RNA extracted using Trizol (Thermo Fisher, #15596-026). 100 µg of extracted RNA were incubated for 2 h at room temperature with rotation in 1/10 volume of 10X biotinylation buffer (Tris-HCl pH 7.4, 10 mM EDTA) and 2/10 Volume of biotin-HPDP (1mg/ml in Dimethylformamide (Thermo Fisher, #21341)).

Growth protocol

Mouse DTCM23/49 XY embryonic stem cell lines (mESCs) were cultured in Knockout Dulbecco’s Modified Eagle Medium (D-MEM, Thermo Fisher, #10829-018) supplemented with 15% fetal bovine serum (FBS, Thermo Fisher, #16000-044), 1% antibiotic penicillin/streptomycin (Thermo Fisher, 15070063), Recombinant mouse LIF protein to a final concentration of 0.01% (Merck, #ESG1107) and 0.06 mM 2-Mercaptoethanol (Thermo Fisher, #31350-010), on 0.1% gelatin-coated cell culture dishes. When confluent, culture was divided in two and passaged 8 times. Five million mESCs of each of these 2 biological replicates were seeded on a 10 cm 0.1% gelatin-coated tissue plate and allowed to grow to 70-80% confluency (approximately 1 day).

Extracted molecule

total RNA

Extraction protocol

RNA was extracted using phenol:chloroform:isoamyl alcohol (Sigma, P3803-400ML). Equal volume of biotinylated RNA and prewashed DynabeadsTM MyOneTM Streptavidin T1 beads (Thermo Fischer, 65601) were added to 2X B&W Buffer (10 mM Tris-HCl pH7.5, 1mM EDTA, 2M NaCl (Thermo Fisher, #65601)) and incubated at room temperature for 15 minutes under rotation. The beads were then separated from the mixture using DynaMagTM-2 Magnet (Thermo Fisher, #12321D) and after removal of supernatant, washed with 1X B&W three times. Biotinylated RNA was recovered from the supernatant after 1 minute incubation with RTL buffer (RNeasy kit, Qiagen, #74104) and purified using the RNeasy kit according to manufacturer’s instructions.
Total RNA libraries were prepared from 10 ng of DNAse treated total RNA and newly transcribed RNA using Ovation® RNA-Seq and sequenced on Illumina HiSeq 2500.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Hundred nucleotides long single-end stranded reads were mapped to mouse ribosomal RNA sequences with STAR V 2.5.0 (Dobin et al., 2013).
Reads not mapping to ribosomal RNA were then aligned to intronic and exonic sequences using STAR and quantified using RSEM (Li and Dewey, 2011).
Rates of synthesis, processing and degradation were independently inferred using biological duplicates at each labelling points using the INSPEcT Bioconductor package Version 1.8.0 (de Pretis et al., 2015) and biotype differences in the average rate across the 3 labeling times.
Genome_build: mm10
Supplementary_files_format_and_content: The processed data table contains the expression levels of transcript exons and introns (Transcripts Per Million, TPM). Values below expression cutoff (TPM < 1) were converted to NAs and excluded in downstream analysis.

Submission date

Mar 16, 2018

Last update date

Mar 21, 2018

Contact name

Jennifer Yihong Tan

E-mail(s)

jennifer.tan@unil.ch

Phone

+4121 692 5472

Organization name

University of Lausanne