GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM6932663

Query DataSets for GSM6932663

Status

Public on Jun 07, 2023

Title

mgm3044-3

Sample type

SRA

Source name

MC2 155

Organism

Mycolicibacterium smegmatis MC2 155

Characteristics

strain: MC2 155
genotype: R25E CarD

Growth protocol

Mycobacterium smegmatis cells were cultured in LB media supplemented with 0.5% (v/v) dextrose, 0.5% (v/v) glycerol, and 0.05% (v/v) tween-80 in 50mL glass flasks shaking at 150rpm at 37 C.

Extracted molecule

total RNA

Extraction protocol

Mycobacterium smegmatis cells were lysed by bead-beating in Trizol reagent (Invitrogen) and then RNA was isolated by chloroform extraction.
RNA samples were treated with DNAse using the TURBO DNA-free kit (Invitrogen). Libraries were prepared with 500ng of total RNA. Ribosomal RNA was blocked using FastSelect reagents (Qiagen) during cDNA synthesis. RNA was fragmented in reverse transcriptase buffer with FastSelect reagent and heating to 94 degrees for 5 minutes, 75 degrees for 2 minutes, 70 degrees for 2 minutes, 65 degrees for 2 minutes, 60 degrees for 2 minutes, 55 degrees for 2 minutes, 37 degrees for 5 minutes, 25 degrees for 5 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 15 cycles using primers incorporating unique dual index tags.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

QC was performed using FastQC version 0.11.8 with default parameters
Adapter trimming and poor quality read filtering and was performed using Trimmomatic version 0.38.0 with the ILLUMINACLIP function targeting Illumina TruSeq3 paired-end adapter sequences and using a Sliding Window approach to trim reads with a quality <=20
Reads were aligned to the M. smegmatis MC2 155 reference genome using HISAT2 version 2.2.1 with default parameters for paired-end reads
Read counts aligning to annotated genes were quantified using featureCounts version 2.0.1
Differential expression analysis was performed using DESeq2 package version 1.30.1 implemented in Rstudio RStudio 2022.02.3+492 "Prairie Trillium" Release for macOS
Assembly: Mycobacterium smegmatis ASM1500v1
Supplementary files format and content: msmeg_countTable.csv - comma separated values table with results of featureCounts quantification of reads aligning to annotated genes
Supplementary files format and content: msmeg_differentialExpression.csv - comma separated values table with the results of differential expression analysis performed using DESeq2

Submission date

Jan 13, 2023

Last update date

Jun 07, 2023

Contact name

Dennis Zhu

Organization name

Washington University

Lab

Stallings Lab

Street address

660 South Euclid Avenue