|
| Status |
Public on May 02, 2023 |
| Title |
Dataset D8 lib5 |
| Sample type |
SRA |
| |
|
| Source name |
Whole bacteria
|
| Organism |
Staphylococcus aureus |
| Characteristics |
tissue: Whole bacteria
cell line: JE2
cell type: Bacteria
genotype: JE2, JE2 PGbaA-L-sGFP, JE2 gbaA PGbaA-L-sGFP, JE2 Phom-sGFP
treatment: TSB
|
| Treatment protocol |
After growth as described, most bacteria were not subjected to further treatment.
|
| Growth protocol |
Bacteria were streaked out onto agar from frozen stocks overnight then grown overnight in TSB. Samples were then diluted back to grow in the medium and to the densities indicated. There was an initial "back-dilution" step prior to harvesting to improve population homogeneity.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Samples were fixed and processed according to the protocol of Blattman et al. (PMID: 32451472). Briefly, cells were fixed overnight with 4% formaldehyde, permeabilized with 50% ethanol followed by enzymatic cell wall digestion (100 µg/ml lysostaphin, 15 min), then treated with DNase to remove genomic DNA.
Reverse transcription and split-pool barcode ligation were carried out in situ in fixed cells according to the protocol of Blattman et al. (PMID: 32451472). After barcoding, cells were separated into aliquots of ~20,000 cells for further processing. After lysis and reversal of crosslinking, second strand synthesis was performed using the NebNext Second Strand Synthesis module (New England Biolabs) and tagmentation was carried out using the EZ-Tn5 transposase (Lucigen), before final amplification with multiplex indexing primers.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
S. aureus grown to an OD600 of ~0.1 from JE2 WT and gbaA mutant, and sGFP reporter strains
D8_metadata.txt
|
| Data processing |
All analysis was performed using the Tavazoie lab computational pipeline (https://tavazoielab.c2b2.columbia.edu/PETRI-seq/PETRI_Seq_Computational.pdf).
Initial QC was performed using FastQC then unique molecular identifiers were extracted with UMI tools. Initial demultiplexing by cell barcode was performed iteratively using Cutadapt.
After selecting barcodes associated with the highest number of reads, reads from individual barcodes were aligned to the S. aureus USA300-FPR3757 reference genome using BWA.
Numbers of reads mapping to individual genes were quantified using FeatureCounts before counts were collapsed into unique reads based on UMI and used to make the final counts matrix.
Sample annotations were extracted from the number of barcode 1, since this corresponds to the way samples were added in the initial plate during reverse transcription.
Assembly: S. aureus reads were aligned to the USA300_FPR3757 reference assembly (GCF_000013465.1).
Supplementary files format and content: Counts matrices: gzip-compressed tab-separated text files of counts by genes (columns) by cell barcode (rows) ("_counts.txt.gz")
Supplementary files format and content: Metadata files: tab-separated files containing library and sample information for each cell barcode ("_metadata.txt").
|
| |
|
| Submission date |
Mar 09, 2023 |
| Last update date |
May 02, 2023 |
| Contact name |
Itai Yanai |
| E-mail(s) |
itai.yanai@nyulangone.org
|
| Organization name |
NYU Langone Health
|
| Street address |
435 East 30th Street
|
| City |
New York City |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
| |
|
| Platform ID |
GPL24034 |
| Series (1) |
| GSE217715 |
A quantitative model for the transcriptional landscape of the bacterial cell cycle |
|
| Relations |
| BioSample |
SAMN33706341 |
| SRA |
SRX19625908 |
