|
| Status |
Public on Feb 26, 2024 |
| Title |
Mock riboseq, 15min, rep2 |
| Sample type |
SRA |
| |
|
| Source name |
bacteria
|
| Organism |
Lactococcus lactis |
| Characteristics |
cell type: bacteria
library prep: Ribo-seq
time: 15 minutes
treatment: mock
|
| Treatment protocol |
To achieve a multiplicity of infection (MOI) of ~10, 12 millilitres of phage sk1 in SM buffer (100 mM NaCl, 50 mM Tris HCl, 10 mM MgSO4) with a titre of 5x1010 per mL were added to the two test samples. The same volume of SM buffer alone was added to the two mock samples at the same time. At 2-, 5-, and 15-min post infection, to stall translating ribosomes, chloramphenicol (100 µg/mL) was added for 2 min prior harvesting.
|
| Growth protocol |
Two hundred millilitres of M17 broth supplemented with 5 g/litre glucose and 10 mM CaCl2 were inoculated with overnight culture to OD600nm of ~0.05 and incubated at 30°C with-out agitation to an OD600nm of 0.4-0.43.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were pelleted by centrifugation; pellets were frozen and pulverized in Retsch Mixer Mill 400. 25 AU of lysate were supplemented with 5 mM CaCl2 and digested with 1500 U of Micrococcal nuclease (Roche) at 25°C for 1 hour with agitation at 1400 rpm.
Following end repair, pre-adenylated linkers bearing unique molecular identifiers (UMIs) and sample barcodes were ligated to footprints. rRNA removal was done with Ribo-Zero Magnetic kit for Gram-positive bacteria (Epicentre) according to the manufacturer’s protocol. Single-stranded cDNA was circularized and then amplified by 4 rounds of PCR. Deep sequencing was performed with the Illumina HiSEQ4000.
Other:Ribo-seq
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
These reads have being processed. This includes removal of the adapter sequence with Cutadapt. The multiplexed reads were then binned into individual samples based on the on the barcode. Next the 7 nucleotide UMI were used to remove duplicate sequences. Finally reads that mapped to rRNA or tRNA with Bowtie (version 1.2.3) were discarded.
Reads were mapped to the genomes of L. Lactis subsp. cremoris NZ9000 (accession number NC_017949.1) and SK1 (AF011378.1) with Bowtie, using -m 1 parameter.
Supplementary files format and content: tab-delimited text files include raw mapped reads to each gene for each Sample
|
| |
|
| Submission date |
May 08, 2023 |
| Last update date |
Feb 26, 2024 |
| Contact name |
Martina Yordonova |
| Organization name |
University College Cork
|
| Department |
School of Biochemistry and Cell Biology
|
| Street address |
Western road
|
| City |
Cork |
| ZIP/Postal code |
T12 XF62 |
| Country |
Ireland |
| |
|
| Platform ID |
GPL23778 |
| Series (1) |
| GSE231968 |
Ribosome profiling experiment characterising early response of Lactococcus Lactis infected in sk1 bacteriophage |
|
| Relations |
| BioSample |
SAMN35004583 |
| SRA |
SRX20261078 |
