|
| Status |
Public on Apr 20, 2020 |
| Title |
rsmAF_B: PAO1 ∆rsmAF RNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells grown to OD 0.5
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
genotype: PAO1 with deletion of rsmA and rsmF genes
tissue: mid-logarithmic phase culture
|
| Growth protocol |
Bacterial cultures were started from single colonies, grown overnight, and back-diluted in LB media
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted via Direct-Zol RNA Miniprep Kit (Zymo Research)
Illumina cDNA libraries were generated using a modified version of the RNAtag-Seq protocol. 500 ng - 1 µg total RNA was fragments, depleted of genomic DNA, dephosphorylated and ligated to DNA adapters carrying 5'-AN8-3' barcodes of known sequence with a 5' phosphate and a 3' blocking group. Barcoded RNAs were pooled and depleted of rRNA using the RiboZero rRNA deplection kit (EpiCentre). Pools of barcoded RNAs were converted to Illumina cDNA libraries in 2 main steps: (i) reverse transcription of the RNA usinga primer designed to the constant region of the barcode adaptor with addition of an adapter to the 3' end of the cDNA by template switching using SMARTScribe (Clonetech); (ii) PCR amplification using primers whose 5' ends target the constant region of the 3' or 5' adaptors and whose 3' ends contain the full Illumina P5 or P7 sequences.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Sequencing reads from each pooled sample were demulitplexed based on their associated barcod sequence using custom scripts; allowing up to 1 mismatch in the barcode, provided this did not make assignemtn of the read to a different barcode possible. Barcode sequencese were subsequently trimmed from the first read as were terminal G's from the second read that may have been added by SMARTScirbe during template switching.
Reads were aligned to the PAO1 reference genome (NCBI RefSeq NC_002516) using BWA
Read counds were assigned to the genes and other genomic features using custom scripts
Differential expression analysis was conducted with DESeq2.
For visualization, the bam alignment files from samples 1A, 2C and 3B were sorted using samtools (version 1.10).
The sorted reads from samples 1C, 2C, and 3B were split by strand and convereted to the bedgraph file format using bedtools (version 2.92.2).
The read depth in samples 2C and 3B were normalized to the total number of mapping fragments in sample 1A using custom R scripts.
The normalized bedgraph files from Samples 1C, 2C, and 3 B were converted to the bigwig file format using the bedgraphToBigWig executable (downloaded from the USCS database) for visualization in a genome brower.
Genome_build: NC_002516.2
Supplementary_files_format_and_content: bgwig files were generated using the bedgraphtobigwig script package from UCSC; Scores represent normalized RNASeq read density
|
| |
|
| Submission date |
Feb 13, 2020 |
| Last update date |
Apr 20, 2020 |
| Contact name |
Michael J Gebhardt |
| Organization name |
University of Iowa
|
| Department |
Department of Microbiology and Microbiology
|
| Lab |
Gebhardt
|
| Street address |
431 Newton Road, 200A EMRB
|
| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
| |
|
| Platform ID |
GPL23999 |
| Series (1) |
| GSE138338 |
Widespread targeting of nascent transcripts by RsmA in Pseudomonas aeruginosa |
|
| Relations |
| BioSample |
SAMN14095065 |
| SRA |
SRX7718981 |
