|
| Status |
Public on Feb 25, 2019 |
| Title |
RIBOT_ribo_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Wild-type strain with pRibo-T plasmid cured of plasmid carrying WT rRNA
|
| Organism |
Escherichia coli |
| Characteristics |
strain/variation: SQ171 (+pRibo-T)
|
| Growth protocol |
The cultures of SQ171 (WT) and Ribo-T cells were grown overnight in LB medium supplemented with ampicillin (100 mg/ml) and spectinomycin (50 mg/ml). Cultures were diluted 100-fold into three conical baffled 1-litre flasks containing 100 mL of LB supplemented with the same antibiotics and with 0.2% glucose. Cultures were grown at 37 °C with vigorous shaking.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
When the culture density reached A600 of ∼0.5 cells were harvested by rapid filtration, frozen in liquid nitrogen, and processed for ribosome profiling as described in [Becker, A.H., Oh, E., Weissman, J.S., Kramer, G. & Bukau, B. Selective ribosome profiling as a tool for studying the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes. Nat Protoc 8, 2212-39 (2013)]. For the preparation of RNA-seq samples, total RNA was isolated from the cell lysates, short RNA and rRNA were subtracted using Ambion MEGAclear kit and MICROBexpress bacterial mRNA enrichment kit, respectively, and RNA was fragmented using the RNA Fragmentation Reagents kit from Ambion.
The library for next-generation sequencing was prepared using RNA fragments ranging in size from 20 to 45 nt. During data analysis, the first position of the P site codon was assigned at 15 nt away from the 3’ end of the read.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Evolved fast growth strain
|
| Data processing |
Library strategy: Ribo-seq
The adapter sequence (located in adapters.fasta) was removed (when found) from the 3′ end of all reads using Cutadapt
Lanes were demultiplexed into constituent samples using Cutadapt, and concatenated together using 'cat' to form sample specific .fastq.gz files. (NOTE: These are the provided .fastq.gz files for each sample). Reads without barcodes were discarded from all further analysis.
For subsequent analysis, 5 nucleotides were removed from the 3' terminus of each read using Cutadapt
The 5' adapter sequence (found in adapters.fasta) was removed (when found) using Cutadapt
2 nucleotides were removed from the 5' terminus of each read using Cutadapt
RNA-seq reads only were aligned using Kallisto to a transcript index built from U00096.3 with "-k 17" and analyzed for differential expression using Sleuth resulting in the RNAseq_stat_summary.csv file
For ribosome profiling and translation efficiency analysis, both Ribo-seq and RNA-seq reads were mapped to U00096.3 using HISAT2 (--no-spliced-alignment flag) to create .sam and subsequently .bam files
Fully reproducible bioinformatic details with code can be found at: https://github.com/adamhockenberry/ribo-t-sequencing
Genome_build: genbank id: U00096.3
Supplementary_files_format_and_content: For both sample types (Ribo-seq and RNA-seq), the primary HISAT2 alignments were mapped to the 3' end position of the read to create initial .wig files for visualization and gene-level quantification. After visual inspection an offset of 16 nt was applied to align Ribo-seq data to ribosomal P-sites. The same offset/analysis was applied to RNA-seq for comparison/consistency. RPKM for each CDS was calculated for each sample's Ribo-seq and RNA-seq data from these .wig mappings. Statistical summaries/comparison of Ribo-seq and Translation Efficiency (TE) differences can be found in TE_stat_summary.csv
|
| |
|
| Submission date |
Jan 07, 2019 |
| Last update date |
Feb 25, 2019 |
| Contact name |
Adam John Hockenberry |
| E-mail(s) |
adam.hockenberry@utexas.edu
|
| Organization name |
The University of Texas at Austin
|
| Department |
Department of Integrative Biology
|
| Lab |
Institute for Cellular and Molecular Biology
|
| Street address |
2500 Speedway, A4800, MBB 3.232
|
| City |
Austin |
| State/province |
TX |
| ZIP/Postal code |
78712 |
| Country |
USA |
| |
|
| Platform ID |
GPL21222 |
| Series (1) |
| GSE119454 |
Assembly and functionality of the ribosome with tethered subunits |
|
| Relations |
| BioSample |
SAMN10711896 |
| SRA |
SRX5223012 |
