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Sample GSM1490116

Query DataSets for GSM1490116

Status

Public on Dec 25, 2014

Title

LPS-20min rep 1

Sample type

SRA

Source name

Bone marrow

Organism

Mus musculus

Characteristics

source mice: C57BL/6
age: 8 weeks
gender: male
cell type: bone marrow derived macrophage
treatment: 100 ng/ml LPS
library strategy: GRO-Seq
time: 20 min

Treatment protocol

The primary macrophages were plated for experiments in macrophage serum-free media (Invitrogen) prior to treatment with 100 ng/ml LPS (Sigma-Aldrich) for the time indicated, without or with 1µM Dexamethasone (Sigma-Aldrich).

Growth protocol

All primary macrophages were differentiated from bone marrow isolated from an isogenic C57 background as described previously (Barish et al. 2010)

Extracted molecule

total RNA

Extraction protocol

Nuclei were isolated from primary macrophages and subjected to nuclear run-on in the presence of BrUTP. BrUTP incoorporated nascent RNAs were isolated using anti-BrU antibodies (Santa Cruz, sc-32323 AC) for the construction of sequencing libraries.
GRO-Seq library generation was carried out as described previously (Hah et al. 2013) from two biological replicates of mouse primary macrophages treated with LPS alone or with LPS and DEX as indicated. GRO-Seq libraries were sequenced on an Illumina HiSeq2000 sequencer to a depth of over 50 million reads per library.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

nascent RNA
bone marrow derived macrophage treated with LPS-20min rep 1

Data processing

Basecalls performed using CASAVA version 1.7
PolyA stretches at the end of the reads were clipped using HOMER v4.7 (command: homerTools trim -3 AAAAAAAAAAAA fastqfile)
Trimmed reads were aligned to the mouse genome (mm9) using bowtie2 v2.0.5. Only reads with a single best alignment were kept for downstream analysis.
Downstream analysis of GRO-Seq data was carried out using HOMER (v4.6, http://homer.salk.edu/homer/). This includes calculation of gene expression FPKM levels, identification of transcripts, generation of Genome Browser bedGraphs, etc. Gene expression values were generated by HOMER using RefSeq gene models, and are calculated across the entire gene body (exons+introns).
Genome_build: mm9
Supplementary_files_format_and_content: Text (tab-delimited) file containing FPKM for all experiemnts. Columns: 1) RefSeq ID, 2) chromosome, 3) start, 4) end, 5) strand, 6) length, 7) copies in genome, 8) annotation, 9 on) experiment FPKM

Submission date

Aug 27, 2014

Last update date

May 15, 2019

Contact name

Christopher Benner

E-mail(s)

cbenner@ucsd.edu

Organization name

University of California, San Diego (UCSD)

Department

Medicine