GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5356866

Query DataSets for GSM5356866

Status

Public on Sep 22, 2021

Title

Lung-Mtb-singles 2

Sample type

SRA

Source name

Lung

Organism

Oryctolagus cuniculus

Characteristics

strain: NZW
agent: Mycobacterium tuberculosis as single cells
tissue: Lung
time point: 24 hrs post infection

Extracted molecule

total RNA

Extraction protocol

Total RNA from uninfected, Mtb-SC and -AG-infected rabbit lungs was isolated (n=3) at T=24 h time point. Briefly, portions of frozen lung tissue (0.5g) were thawed in the presence of 10x volume (wt/vol) of TRIzol reagent (Life Technologies, Grand Island, NY, USA) and homogenized on ice. RNA was extracted once with 0.1 volumes (vol/vol) of BCP solution (Molecular Research Center Inc, Cincinnati, OH, USA), and the aqueous phase, containing RNA, was mixed with an equal volume of 70% ethanol and passed through mini spin columns from the Qiagen RNeasy Mini Kit (Qiagen Inc, Germantown, MD, USA). Following on-column digestion of the contaminating DNA using DNaseI, RNA was purified and eluted with sterile water, as described by the manufacturer. The quality and quantity of the purified RNA were assessed by using the NanoDrop instrument (NanoDrop products, Wilmington, DE, USA), and by Bioanalyzer (Agilent Technologies Inc., Santa Clara, CA).
RNAseq experiment was performed by Lexogen (Lexogen, Inc, Greenland, NH, USA). Briefly, cDNA libraries were prepared using Lexogen QuantSeq 3′mRNA-Seq Library Prep Kit FWD for Illumina using standard protocols (Lexogen, Inc, Greenland, NH, USA), and sequencing was performed with NextSeq protocol. The quality of sample reads, mapping, alignment, and read counts were performed using the STAR (Spliced Transcripts Alignment to a Reference) tool and the RSEQC quality control package (Lexogen, Inc, Greenland, NH, USA). After removing genes without reads in all the tested samples, data normalization and differential expression of transcripts were performed with DESeq2 from the Bioconductor package implemented in R. The output data were filtered to select transcripts with Benjamini-Hochberg adjusted p <0.05 using FASTQ/MULTIQC tool (Lexogen, Inc, Greenland, NH, USA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

SC-Group-2

Data processing

QuantSeq FWD followed by Trimming using reference 'polyA' and "truseq"
The STAR aligner (Spliced Transcripts Alignment to a Reference) is Lexogens aligner of choice for aligning reads which were generated in a QuantSeq FWD or QuantSeq REV experiment.
The STAR aligner aligns reads directly to a reference genome via the utilization of a pre-generated index of the reference. A reference consists of sequences in FASTA format and, optionally, information about genes and transcripts stored in a GFF/GTF file. The reads are provided in a text file in the FASTQ format, which is then aligned by the tool. The results of this alignment are the aligned reads in SAM or binary SAM (BAM) format, optionally the unmapped reads in SAM/BAM format and mapping statistics for the alignment.
The STAR aligners key feature is that it maps reads directly against the reference in notable short time, with an inherent drawback of requiring a relatively high amount of short term memory (RAM) compared to other alignment tools to do so.
The differential expression analysis was performed with DESeq2. DESeq2 is a bioconducter package implemented in R.
Genome_build: OryCun2.0 (GCA_000003625.1)
Supplementary_files_format_and_content: There are 3 groups; Group-1=Singles; Group-2=Aggregates; Group-3=Normal (control). Group 1 and 2 were normalized to Group-3 (control) and the resulting data (log2 fold change) is presented as Differentially Expressed list of entities in Excel file

Submission date

Jun 04, 2021

Last update date

Sep 22, 2021

Contact name

Selvakumar Subbian

E-mail(s)

subbiase@njms.rutgers.edu

Phone

9738543226

Organization name

Rutgers Biomedical and Health Sciences, Rutgers The State University of NJ