|
| Status |
Public on May 22, 2014 |
| Title |
EHEC_HTF1 |
| Sample type |
SRA |
| |
|
| Source name |
whole cells_His-FLAG tagged Hfq
|
| Organism |
Escherichia coli O157:H7 str. Sakai |
| Characteristics |
media: MEM-HEPES
rna chaperone hfq: His-FLAG tagged
purification strategy: ANTI-FLAG M2 affinity gel (Sigma-Aldrich, cat# A2220); Ni-NTA resin
strain: Sakai
|
| Treatment protocol |
Cells were crosslinked with 1800mJ of UV-C and collected by centrifugation. Cell were pelleted, resuspended in PBS, and divided into 1g aliquots that were snap frozen in liquid nitrogen.
|
| Growth protocol |
Strains were intially grown in LB for 16hrs and then diluted (1/100) into appropriate media and grown to and OD600 of ~0.8 at 37 degrees C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were lyzed by vortexing with zirconia beads in lysis buffer. Hfq.RNA complexes were purified over M2 anti-FLAG resin, trimmed with RNase A/T1 and repurified under denaturing conditions using Ni-NTA resin. RNA.Hfq complexes were seperated by SDS-PAGE, transferred to a nitrocelluose membrane and extracted. Protein was removed by Prrteinase K digestion and RNAs purified by phenol:chloroform extraction.
Extracted RNAs were converted to cDNA and PCR amplified. PCR products between ~150 and 250 bp were gel extracted and submitted for Solexa sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
RIP-Seq
EHEC hfq::HTF1
|
| Data processing |
barcodes3.pl M=0 barcode_list.txt input.fq. Custom script to demultiplex fastq file.
fastq_preprpcessing.sh -Q 33 -L TGGAATTCTC -M 15 NNNbarcode_input.txt. Custom script to clip linkers and apply quality filters. Based on fastx toolkit
novoalign -f NNNbarcode_input.fq -d Saki.novoindex -h 150 -F STDFQ (or ILFMQ) -r Random. Map reads to E. coli O157:H7 genome using novoalign
pyReadCounters.py -f input.novo --gtf Sakai_UCSC_1.0.gtf. Custom program used to generate hit tables and plots of read density. From the pyCRAC software package (sandergranneman.bio.ed.ac.uk/Granneman_Lab/pyCRAC_software.html).
novo2pws.awk input.novo. Mapped reads were alternatively analysed by converting to PWS format (tab-delimited file with chromosome, position, strand, read, substitution and deletion information at each nucleotide position)
Genome_build: The reads generated from E. coli O157:H7 str. Sakai were mapped against UCSC microbial genome Escherichia coli O157H7 29/3/2000 assembly
Genome_build: The reads generated from E. coli K12 str. MG1655 were mapped against E. coli K12 str. MG1655 genome (NC000913.2)
Supplementary_files_format_and_content: GTF. Hashed header contains overall information including input files and processing commands. Number of mapped reads are given in column 6. The final semi-colon seperated column of "attributes" gives the position of deletions (D) and substitutions (S).
|
| |
|
| Submission date |
Apr 16, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jai Justin Tree |
| E-mail(s) |
j.tree@unsw.edu.au
|
| Phone |
+61 2 938 59142
|
| Organization name |
University of New South Wales
|
| Department |
School of Biotechnology and Biomolecular Sciences
|
| Lab |
Tree lab
|
| Street address |
Rm s110 Bldg F25, UNSW, Gate 11 Botany St
|
| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2033 |
| Country |
Australia |
| |
|
| Platform ID |
GPL17025 |
| Series (2) |
| GSE46118 |
UV-crosslinking and high throughput sequencing of cDNAs (CRAC) of the RNA chaperone Hfq in enterohaemorhaggic E. coli |
| GSE46120 |
Small RNAs and RNA chaperone Hfq in enterohemorrhagic E. coli |
|
| Relations |
| BioSample |
SAMN02046641 |
| SRA |
SRX266376 |
