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Status |
Public on Jun 07, 2023 |
Title |
csm106-1 |
Sample type |
SRA |
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Source name |
MC2 155
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Organism |
Mycolicibacterium smegmatis MC2 155 |
Characteristics |
strain: MC2 155 genotype: K125E CarD
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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.
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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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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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
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Submission date |
Jan 13, 2023 |
Last update date |
Jun 07, 2023 |
Contact name |
Dennis Zhu |
Organization name |
Washington University
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Lab |
Stallings Lab
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Street address |
660 South Euclid Avenue
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City |
St. Louis |
State/province |
MO |
ZIP/Postal code |
63110-1093 |
Country |
USA |
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Platform ID |
GPL28006 |
Series (1) |
GSE222815 |
Transcription regulation by CarD in mycobacteria is guided by basal promoter kinetics |
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Relations |
BioSample |
SAMN32727277 |
SRA |
SRX19026467 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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