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Sample GSM2233347

Query DataSets for GSM2233347

Status

Public on Dec 01, 2016

Title

RNA shKdm5ABC 4 hour exp 1

Sample type

SRA

Source name

3T3-L1_RNA shKdm5ABC 4 hour

Organism

Mus musculus

Characteristics

cell line: 3T3-L1
cell type: Preadipocyte cell line
transduced with: shKdm5ABC
time point: 4 hours post induction
molecule subtype: polyA-enriched mRNA

Treatment protocol

Two days post confluency (day 0), 3T3-L1 cells were induced to differentiate using a standard cocktail of adipogenic inducers (dexamethasone, insulin, 3-isobutyl-1-methylxanthine, and fetal bovine serum). Two days post induction (day 2), medium was changed to DMEM supplemented with fetal bovine serum and insulin. Cells were maintained in DMEM supplemented with fetal bovine serum for the remainder of the differentiation. For knock down experiments, pre-confluent 3T3-L1 cells were transduced 4 days before induction of differentiation by incubation for 24 hours at 37°C with medium containing lentiviral constructs (shKDM5A/B/C or control shLuc) and 6 μg/mL polybrene (Sigma). After 24 hours the medium was changed to normal growth medium.

Extracted molecule

polyA RNA

Extraction protocol

Total RNA from 3T3-L1 cells was purified using TRIzol (Invitrogen) according to the manufacturer’s instructions. RNA was treated and the integrity of the RNA was confirmed using a Bioanalyzer 2100 (Agilent Technologies). 4 ug of total RNA was polyA-selected, and subsequently subjected to fragmentation and cDNA-synthesis using the TruSeq RNA Sample Prep kit v2 (Illumina). cDNA-seq libraries were constructed according to the manufacturer's instructions (Illumina) as described in (Nielsen R, Mandrup S, Methods in Enzymology 2014, Vol. 537, pp. 261-279).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Description

processed data file: RNA_all_genes_log2_tags_kb.txt

Data processing

RNA reads were aligned to the mouse reference genome (version mm9) using Bowtie2 (version 0.12.8) (Langmead and Salzberg 2012, Nat Met 9: 357-359) with standard parameters. Splice-junction reads were handled by creation of a pseudo-splice genome, similar to the strategy utilized in RSEQTools (Habegger et al. 2011, Bioinformatics 27: 281-283). Reads were filtered post-alignment for a MAPQ score greater than or equal to 30. The number of exon reads for all RefSeq genes were counted using Subread (Liao et al. 2013, Nucleic Acids Res 41: e108) and differential expression analyses were performed using DESeq2 (FDR <0.05, paired analysis) (Love et al., 2014, Genome Bio. 15: 550).
Genome_build: mm9
Supplementary_files_format_and_content: H3K4me3_promoter_peaks_tags.txt: Table containing promoter H3K4me3 binding sites, including normalized tag counts for H3K4me3 libraries
KDM5A_all_peaks_tags.txt: Table containing all identified KDM5A binding sites, including normalized tag counts for KDM5A libraries
KDM5C_all_peaks_tags.txt: Table containing all identified KDM5C binding sites, including normalized tag counts for KDM5C libraries
RNA_all_genes_log2_tags_kb.txt: Table containing gene expression data for all RNA-seq libraries (reported as log2(tags/kb))

Submission date

Jul 14, 2016

Last update date

May 15, 2019

Contact name

Ann-Sofie Bøgh Brier

E-mail(s)

a.boegh@me.com

Phone

+4522969284

Organization name

University of Southern Denmark, Odense