NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM6932660 Query DataSets for GSM6932660
Status Public on Jun 07, 2023
Title mgm3043-4
Sample type SRA
 
Source name MC2 155
Organism Mycolicibacterium smegmatis MC2 155
Characteristics strain: MC2 155
genotype: WT 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
City St. Louis
State/province MO
ZIP/Postal code 63110-1093
Country USA
 
Platform ID GPL28006
Series (1)
GSE222815 Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics
Relations
BioSample SAMN32727282
SRA SRX19026462

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap