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Sample GSM3866807

Query DataSets for GSM3866807

Status

Public on Nov 04, 2019

Title

AX4 Non-inf-B

Sample type

SRA

Source name

Cell culture

Organism

Dictyostelium discoideum AX4

Characteristics

strain: AX4
infection: non-infected ctrl

Treatment protocol

Experimental procedure for M. marinum infection samples: the day before infection, BD Falcon tissue culture dishes (100 × 20 mm) were seeded with D. discoideum AX2 cells and grown without antibiotics overnight to 2x10^7 cells per plate. M. marinum were collected and resuspended in HL5-C, bacterial clumps were disrupted by passing through a blunt end 26-gauge needle whereafter bacteria was added to the amoeba at MOI ~200. Non-infected samples were prepared simultaneously and treated identically except for the addition of bacteria. The infection was initiated by centrifugation at 500xg twice for 7 minutes or three times for 5 minutes followed by incubation at 25°C for 50 min post infection to allow for uptake of bacteria. Subsequently, extracellular M. marinum cells were removed by five washes with HL5-C after which the cells were incubated at 25°C. The cells were collected in HL5-C approximately 2 hours after initiation of infection and a subsample was added to the same volume of Soerensen buffer (2g KH2PO4 and 0.27g Na2HPO4 in 1L water, pH 6) + 5mM Sodium azide (Sigma). Samples were analyzed in a BD Accuri C6 flow cytometer in Soerensen buffer + 20 mM sorbitol. Experimental procedure for L. pneumophila infection samples is described in Li et al., 2009 (DOI:10.1016/j.chom.2009.08.005).

Growth protocol

For M. marinum infection samples: Dictyostelium discoideum AX2 cells were cultured axenically at 22°C in 10 ml HL5-C medium without glucose pH 6.4 (Formedium) supplemented after autoclavation with 1% glucose and 100 U/ml Penicillin-Streptomycin (Gibco by Life Technologies). The cells were grown adherent to plastic in BD Falcon tissue culture dishes (100 × 20 mm). Antibiotics were excluded from the growth medium the day before and during infection experiments. Mycobacterium marinum M strain carrying the msp12::gfp plasmid, constitutively expressing GFP, was used in the infection experiments (Hagedorn and Soldati, 2007). Bacteria were grown on 7H10 agar (Difco Middlebrook, BD) containing 10% OADC supplement (oleic acid, albumin, dextrose, catalase; Difco) and 0.5% glycerol, or cultivated in 7H9 broth (Difco Middlebrook, BD) supplemented with OADC, 0.2% glycerol and 0.05% Tween80 (Sigma Aldrich) at 32°C shaking culture with glass beads to decrease bacterial clumping. Both 7H10 agar and 7H9 broth were supplemented with 50 µg/ml kanamycin to maintain the msp12::gfp plasmid. Growth of D. discoideum AX4 and L. pneumophila Lp01 is described in Li et al., 2009 (DOI:10.1016/j.chom.2009.08.005).

Extracted molecule

polyA RNA

Extraction protocol

For M. marinum infection samples, total RNA was isolated by the Ambion PureLink™ RNA Mini kit in combination with TRIzol (Invitrogen), followed by on column DNAse treatment according to the manufacturer’s instructions (Ambion, Invitrogen). For L. pneumophila infection samples RNA samples were prepared by TRIzol extraction.
For M. marinum infection samples, poly(A)-enriched libraries were created using TruSeq RNA Library Prep Kit (Illumina). For L. pneumophila infected samples, libraries were prepared using TruSeq stranded mRNA library preparation kit with polyA selection (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Alignment of poly(A) RNA-seq reads using TopHat v.2.0.13
Read counts performed with featureCounts v 1.6.2 using gene annotations generated Aug. 24th 2018 from www.dictybase.org
Genome_build: D. discoideum (05-13-2009)
Supplementary_files_format_and_content: tab-delimited raw read count tables

Submission date

Jun 10, 2019

Last update date

Nov 04, 2019

Contact name

Erik Holmqvist

E-mail(s)

erik.holmqvist@icm.uu.se

Organization name

Uppsala University

Street address

Husargatan 3, Box 596, Uppsala University, Dept. of Cell and Molecular Biology