|
| Status |
Public on Dec 23, 2020 |
| Title |
PAO1 ∆pilY1 RNA-Seq (AML91) 1 |
| Sample type |
SRA |
| |
|
| Source name |
Bacterial cells grown to OD 0.35
|
| Organism |
Pseudomonas aeruginosa PAO1 |
| Characteristics |
genotype: PAO1 with deletion of pilY1 gene
growth phase: mid-logarithmic phase culture
|
| Growth protocol |
Bacterial cultures were started from single colonies, grown overnight to saturation, and back-diluted in LB media to OD600 ~0.01 and grown with aeration at 250 rpm at 37°C to a final OD600 ~0.35.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trireagent and ethanol precipitation using the method of Goldman, et al. (Mol Cell, 2011. 42(6): p. 817-25.). Note that samples 1-9 were grown together on a single day and sequenced together; samples 10-15 were grown together and sequenced together at a later date.
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 depletion kit (EpiCentre). Pools of barcoded RNAs were converted to Illumina cDNA libraries in 2 main steps: (i) reverse transcription of the RNA using a 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 NovaSeq 6000 |
| |
|
| Description |
Sample 1
dpilY1.1
RNAtag-Seq
5primeRNAseq_PAO1_counts.txt
|
| Data processing |
Sequencing reads from each pooled sample were demultiplexed based on their associated barcode sequence using custom scripts; allowing up to 1 mismatch in the barcode, provided this did not make assignment of the read to a different barcode possible. Barcode sequences 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_002516.2) using BWA.
Read counts were assigned to the genes and other genomic features using custom scripts.
Differential expression analysis was conducted with DESeq2 and/or EdgeR.
For visualization of RNASeq data, the bam alignment files were sorted using samtools (version 1.10), split by strand and converted to the bedgraph file format using bedtools (version 2.92.2).
The read depth in the bedgraphs files were normalized to the sample with the fewest number of mapped reads using custom R scripts.
The normalized bedgraph files were subsequently converted to the bigwig file format using the bedgraphToBigWig executable (downloaded from the UCSC database) for visualization in a genome browser.
Genome_build: NC_002516.2
Supplementary_files_format_and_content: bigWig files were generated using the bedgraphtobigwig script package from UCSC; Scores represent normalized RNASeq read density.
Supplementary_files_format_and_content: 5primeRNAseq_PAO1_counts.tsv: Read counts.
|
| |
|
| Submission date |
Jul 23, 2020 |
| Last update date |
Dec 23, 2020 |
| Contact name |
Andrew M Lippa |
| Organization name |
Boston Children's Hospital
|
| Department |
Pediatrics, Division of Infectious Diseases
|
| Lab |
Dove Lab
|
| Street address |
300 Longwood Ave
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL28916 |
| Series (2) |
| GSE155023 |
H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription [RNA-seq] |
| GSE155025 |
H-NS-like proteins in Pseudomonas aeruginosa coordinately silence intragenic transcription |
|
| Relations |
| BioSample |
SAMN15636532 |
| SRA |
SRX8816661 |
