GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5292347

Query DataSets for GSM5292347

Status

Public on May 13, 2021

Title

PAO3

Sample type

SRA

Source name

choanoflagellate cells

Organism

Monosiga brevicollis

Characteristics

strain: M. brevicollis MX1 ATCC PRA-258
genotype: wild type
treatment: P. aeruginosa conditioned media for 3 hours
tissue: choanoflagellate cells

Treatment protocol

To grow large numbers of cells for RNA-seq, cells were seeded one to two days prior to the experiment in either 3-layer flasks (Falcon; Corning, Oneonta, NY, USA; Cat. No. 14-826-95) or 75 cm2 flasks (Falcon; Corning, Oneonta, NY, USA; Cat. No. 13-680-65), and grown at room temperature. Bacteria were washed away from choanoflagellate cells through two rounds of centrifugation and resuspension in artificial seawater (ASW). To count the cell density, cells were diluted 100-fold in 200 µl of ASW, and fixed with 1 µL of 16% paraformaldehyde. Cells were counted on a hemocytometer, and the remaining cells were diluted to a final concentration of 4×106 choanoflagellate cells/mL. The resuspended cells were divided into 2.5 mL aliquots and plated in 6-well plates prior to treatment. After treatment, cells were transferred to a 15 mL conical and pelleted by centrifugation at 2400 x g for 5 min, flash frozen with liquid nitrogen, and stored at -80°C.

Growth protocol

M. brevicollis were grown in High Nutrient Medium (10% Cereal Grass Media, 2.5% Sea Water Complete Media in artificial sea water) at room temperature

Extracted molecule

total RNA

Extraction protocol

RNA libraries were generated with the Illumina TruSeq® Stranded mRNA Library prep kit (cat # 20020594), using a starting total RNA input of 2-3 µg. To remove contaminating bacterial RNA, samples were first poly-A selected using oligo-dT attached magnetic beads. Following purification, the mRNA was fragmented at 94ºC for 4 minutes, and cleaved RNA fragments were synthesized into cDNA. After an end repair step, UMI adapters (synthesized by IDT) were ligated to the cDNA, and the products were twice purified using AMPure XP beads before amplification. Library quantity was measured using the Quant-iT™ PicoGreen dsDNA Assay kit by Invitrogen (cat # P7589) and a PerkinElmer Victor X3, 2030 Multilabel Reader. Library quality was verified on an Agilent 2100 Bioanalyzer instrument using Agilent High sensitivity DNA kit (cat # 5067-4626) or DNA 1000 kit (cat # 5067-1504). Libraries were pooled, and sequenced on either the Illumina NovaSeq 6000 system with S4 flowcell and XP PE-100 workflow, or on the Illumina NextSeq 550 system with SE-75 workflow.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

+P. aeruginosa rep 3

Data processing

Single-end/paired-end 76 bp read length fastq files were checked for quality using fastqc (v0.11.2) and fastq_screen (v0.11.4)
Reads were quality trimmed using fastq-mcf (ea-utils, v1.05).
Trimmed fastq files were mapped to M. brevicollis genome using TopHat (v2.0.12)
Duplicates were marked using picard-tools (v2.10.10)
Read counts were generated using featureCounts (subread) for coding genes using gtf (Monosiga_brevicollis_mx1.V1.0)
Normalized read counts generated using edgeR
Genome_build: Monosiga brevicollis mx1.V1.0
Supplementary_files_format_and_content: tab delimited txt files that includes normalized read counts for each sample per gene

Submission date

May 12, 2021

Last update date

May 13, 2021

Contact name

Arielle Woznica

E-mail(s)

[email protected]

Phone

2146485627

Organization name

UT Southwestern