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Sample GSM1599743

Query DataSets for GSM1599743

Status

Public on Sep 07, 2015

Title

macrophage (exposed to S. enterica LPS) 38

Sample type

SRA

Source name

bone marrow-derived macrophages

Organism

Mus musculus

Characteristics

cell type: bone marrow-derived macrophages
strain: C57/BL6
hours after exposure (0 for unexposed): 4
beads coated with lps?: No
internalized bead?: No

Treatment protocol

For coating of latex beads, 30 μg of LPS from S.enterica (Sigma, St. Louis, MO) were incubated at 4°C for 12 hours with 1*10^8 of 1μm yellow-green fluorescent polystyrene latex beads (Sigma, St. Louis, MO). Beads were then extensively washed to remove any free LPS. Latex beads (coated or uncoated) were used to stimulate cells at an MOI of 1:1. After allowing 30 minutes for bead uptake, macrophages were washed to remove excess beads

Growth protocol

We prepared cultures of bone marrow derived macrophage cells (BMMs) from 6-8 week old female C57BL/6 mice as previously described (Falk et al., 1988). BMMs were maintained in DMEM (Life Technologies, Carlsbad, CA) supplemented with 20%FBS (Life Technologies, Carlsbad, CA), 5% L-glutamine (Life Technologies, Carlsbad, CA) and 25ng/ml recombinant murine m-CSF (R&D Minneapolis, MN). At 5 days of in vitro culture, macrophages were seeded in 6-well non-tissue culture treated plates (5*10^5 cells per plate).

Extracted molecule

polyA RNA

Extraction protocol

Single cells were sorted into individual wells of a 96 well plate containing 5 µl TCL buffer supplemented with 1% 2-mercaptoethanol (Qiagen, Valencia, CA) according to fluorescent phenotype using FACS. RNA was isolated using 2.2x RNAClean SPRI beads (Beckman Coulter Genomics, Danvers, MA)
To prepare amplified cDNA, we used a slightly modified version of the manufacturer's protocol from the SMARTer Ultra Low RNA Kit ((Clontech, Mountain View, CA) (See linked articule for details). Fragmentation and library constuction were were done using the Nextera XT kit (Illumina, San Diego, CA) according to the manufacturer’s instructions, with all quantities adjusted the ¼ of the recommended volumes.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

Column D2 of prcessed data file
LPS_TPM_table.txt
LPS_Counts_table.txt

Data processing

Alignment and transcript quantification was done using RSEM v1.2.3 (Li and Dewey, 2011) using the following command line options: rsem-calculate-expression –paired-end --calc-ci –estimate-rspd. Genes were quantified using TPM for normalized expression measurements or raw counts for differential expression analysis.
Cells containing fewer than 50,000 mapped reads or having fewer than 4,000 detected genes (TPM > 0) were removed from further ananlysis (but are contained in the attached files).
Genome_build: Reads were aligned to the mouse transcriptome generated using rsem-prepare-reference (default parameters), the Dec. 2011 (GRCm38/mm10) build of the mouse genome, and release 69 of the Ensembl mm10 gene annotations.
Supplementary_files_format_and_content: TPM_table: a table containing TPM values for every ensembl gene(rows) for each single cell (columns). Counts_table: A table containing raw count values for each Ensembl gene (rows) in each single cell (columns)

Submission date

Feb 02, 2015

Last update date

May 15, 2019

Contact name

Deborah T Hung

Organization name

Broad Institute

Department

Infectious Disease Initiative

Lab

Hung lab