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Sample GSM1599701

Query DataSets for GSM1599701

Status

Public on Sep 07, 2015

Title

macrophage (exposed to S. enterica) 188

Sample type

SRA

Source name

bone marrow-derived macrophages

Organism

Mus musculus

Characteristics

cell type: bone marrow-derived macrophages
strain: C57/BL6
time after salmonella exposure (h): 8
phrodo positive?: Yes
gfp positive?: Yes

Treatment protocol

Macrophages were treated with GFP labeled S. enterica (strain SL1344 containing the plasmid pFPV25.1) at an MOI of 1:1. Bacterial cultures were grown overnight in Luria-Bertani (LB) medium at 37°C shaken at 250 rpm, washed with PBS, and stained with pHrodo dye (Life Technologies, Carlsbad, CA) for 1 hour at room temperature in 100mM Sodium Bicarbonate. After the addition of bacteria, cell cultures were spun down for 5 minutes at 250xg. After a 30 minutes incubation, bacteria were removed by washing with media containing 15ug/ml Gentamicin

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 intp 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 G12 of processed data file
s121_p2_TPM_table.txt
s121_p2_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