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| Status |
Public on Jan 10, 2022 |
| Title |
KrrA-FLAG+205#1 |
| Sample type |
SRA |
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| Source name |
Bacterial cells, pelleted
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| Organism |
Bacillus anthracis |
| Characteristics |
strain: Sterne
genotype: {delta}krrA pOS1.PlgtkrrA-FLAG
growth media: LB + 50 µM '205
atomosphere: aerobic
growth temperature: 37°C
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| Treatment protocol |
Bacterial cells were harvested by centrifugation, washed with PBS, subjected to crosslinking using formaldehyde (0.75% final) at room temperature for 10 min, then treated with glycine (125 mM final, pH7.5) at room temperature for 5 min to quench the crosslinking reaction. The cells were subsequently harvested by centrifugation, washed twice with buffer A (50 mM Tris pH 7.4, 150 mM NaCl and 1 mM EDTA), and resuspended in 0.5 ml buffer A amended with 1mM PMSF and 2µl RNase inhibitor followed by sonication for cell lysis and DNA fragmentation.
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| Growth protocol |
Bacteria were grown in LB medium overnight and subcultured at a 1:100 ratio into fresh LB medium. After 6 h of growth at 37°C in the presence or absence of 50 µM ‘205,
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| Extracted molecule |
total RNA |
| Extraction protocol |
The cell lysates (supernatants) were collected after centrifugation. Aliquots of 1% lysates were diluted with buffer A and kept at −80°C to serve as the input control (1% of input RNA). The remaining lysates were incubated with α-FLAG M2 magnetic agarose beads (Sigma, Cat# M8823) on a rotating wheel for 2 h at 4°C for immunoprecipitation. The bead slurry was recovered using a magnetic stand, washed twice with 1ml of high-salt wash buffer (50mM Tris pH7.4, 1 M NaCl, and 1mM EDTA), then washed once with 1 ml of IP wash buffer (50mM Tris pH7.4, 10mM MgCl2, and 0.2% Tween-20). Aliquots of 5% beads were saved for immunoblotting to examine the immunoprecipitation efficiency. The remaining beads were subjected to DNAase treatment and the protein–RNA complexes were eluted with 3X FLAG peptide according to the manufacturer’s protocol. All samples including 1% input RNA samples were incubated in buffer B (50mM Tris pH7.4, 10mM DTT, 1% SDS, 1 mg ml-1 protease K, and 5mM EDTA) at 70oC for 45 min to reverse crosslinking. Enriched RNAs were extracted using Trizol, precipitated using isopropyl alcohol with linear acrylamide, washed twice with 75% ethanol, and quantified using a NanoDrop spectrophotometer.
Library construction was performed using NEBNext Ultra II RNA Library Prep Kit for Illumina and NEBNext Multiplex Oligos for Illumina
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RIPed RNA
KrrA-FLAG+205
4590-HP-15
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| Data processing |
Raw data files (.fastq) were imported into the CLC Genomics Workbench software (Version 20.0.1) platform for analysis. The Illumina paired reads option was chosen for import.
Imported reads were trimmed to remove adapters using CLC Genomics Workbench
To remove rRNA reads, all paired reads were aligned to a sequence list containing all rRNA sequences for each corresponding bacterial strain. Unmapped (non-rRNA) reads were collected and used for subsequent analysis. Steps were performed using CLC Genomics Workbench. The refrence genome NC_005945 was used for mapping.
Expression values (as RPKM) for Gene Tracks were caculated using the "RNA-Seq Analysis" function in CLC Genomics Workbench. For cross-comparison between strain a 'consensus genome' was generated by BLASTing every CD630 gene agains each CD196 gene and generating a gene track of the resulting consensus sequences.
Comparison of samples was performed using the "Differential Expression in Two Groups" function in CLC Genomics Workbench
Output was exported to MS Excel and manually curated if neccesary
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| Submission date |
Jan 07, 2022 |
| Last update date |
Jan 10, 2022 |
| Contact name |
Hualiang Pi |
| E-mail(s) |
hualiangpi@gmail.com
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| Organization name |
VUMC
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| Street address |
1161 21st Avenue South
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| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
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| Platform ID |
GPL31186 |
| Series (2) |
| GSE193211 |
A Bacillus anthracis RNA binding protein post-transcriptionally regulates two component signaling through RNA turnover [RIP-Seq] |
| GSE193212 |
A Bacillus anthracis RNA binding protein post-transcriptionally regulates two component signaling through RNA turnover |
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| Relations |
| BioSample |
SAMN24720873 |
| SRA |
SRX13654318 |
| 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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