|
| Status |
Public on Sep 16, 2019 |
| Title |
70S_RPF_Wt |
| Sample type |
SRA |
| |
|
| Source name |
70S_Wt.gz
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
strain background: K-12 MG1655
genotype/variation: wild type
molecule subtype: 70S subunit ribosome protected fragment
|
| Treatment protocol |
Cells were snap chilled using ice and crosslinked with formaldehyde. Glycine was added to arrest the crosslinking after which the cells were pelleted and lysed by freeze thaw method followed by clarification of the lysate. Lysate was loaded on a 10-20 % sucrose gradient and the ribosome pellets were resuspeded in fresh lysis buffer
|
| Growth protocol |
Wt or mutants were grown at 25 °C in 2 litres of LB medium. The culture was allowed to grow till OD600 reached 0.3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Ribosomes bound to mRNA were relieved using MNase.The treated sample was again sepeated into 30S and 70S fractions using ultracentrifugation on a 10-40% sucrose gradient. The 30S and 70S fractions were decrosslinked and ribosome protected fragments were size selcted for 10-80 nucleotide on a denaturing Urea PAGE.
Libraries were prepared strictly according to the manufacturers recommendations using the TruSeq Small RNA Sample Preparation kit (Illumina, U.S.A)
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Reads were filtered by removing reads with Phred score <20 using fastq_quality_trimmer
Trimming of 3’-end adapter sequences [5’-TGGAATTCTCGGGTGCCAAGGAACTC-3’] using Cutadapt-1.5
Alignment with E. coli reference genome NC_000913.3 using STAR 2.5.3 to remove reads mapping to genes for rRNA, tRNA, sRNA and other non-coding RNAs
Filtered reads aligned to E. coli reference genome using STAR 2.5.3 with spliced alignment turned off (--alignIntronMax 1), forced end to end alignment (--alignEndsType EndToEnd) and allowing 8 mismatches per 100 nt (--outFilterMismatchNoverLmax 0.08)
Sam to Bam conversion was accomplished using Samtools-1.4.1. Metagene analysis for mapping the reads to START and STOP codons was performed by employing custom written shell scripts utilizing Samtools-1.4.1.
Genome_build: NC_000913.3
Supplementary_files_format_and_content: .txt files with read counts in tabular format with geneIDs and counts per sample.
|
| |
|
| Submission date |
Nov 23, 2018 |
| Last update date |
Sep 16, 2019 |
| Contact name |
B Anand |
| E-mail(s) |
banand@iitg.ac.in
|
| Organization name |
Indian institute of technology guwahati
|
| Department |
Biosciences and Bioengineering
|
| Lab |
B.Anand
|
| Street address |
North guwahati
|
| City |
Guwahati |
| State/province |
Assam |
| ZIP/Postal code |
781039 |
| Country |
India |
| |
|
| Platform ID |
GPL21117 |
| Series (1) |
| GSE122870 |
Translation complex profiling for bacteria [Ribo-seq] |
|
| Relations |
| BioSample |
SAMN10472883 |
| SRA |
SRX5058105 |
