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Status |
Public on Jun 05, 2016 |
Title |
Laublab_RNAseq_NA1000_PYE |
Sample type |
SRA |
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Source name |
Caulobacter crecentus CB15
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Organism |
Caulobacter vibrioides CB15 |
Characteristics |
developmental stage: mixed population, exponential in PYE genotype: wild type restriction enzyme: NA
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Treatment protocol |
Mixed population of cells growing in PYE (OD 600=0.4) were collected, washed and incubated in M2 salts for 90 min before being snap-frozen for RNA extraction by Hot-Trizol
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Growth protocol |
Caulobacter crescentus were grown as described previously in Skerker et al., 2005 (PMID: 16176121). When appropriate, media were supplemented with antibiotics at the following concentrations (liquid/solid media) [ug/ml]): chloramphenicol (1/2), kanamycin (5/25), oxytetracycline (1/2), spectinomycin (25/100). Synchronizations were performed on mid-exponential phase cells using Percoll (GE Healthcare) and density gradient centrifugation as described previously in Jones et al., 2001 (PMID: 11283290). After synchronization, swarmer cells were fixed formadehyde for chromosome conformation capture and Hi-C analysis. For antibiotic treatments, swarmer cells were incubated with 50 ug/mL chloramphenicol (final concentration) for 30 minutes before fixing with formadehyde. For starvation experiment, swarmer cells were released into M2 salts for 90min before fixing with formadehyde.
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Extracted molecule |
total RNA |
Extraction protocol |
Hi-C experiments were performed using either BglII or NcoI according to previous publications (Umbarger MA. 2012. Chromosome conformation capture assays in bacteria. PMID: 22776362 and Lieberman-Aiden, E. et al., 2009. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. PMID: 19815776). RNA were extracted using Hot Trizol. Detailed protocols are listed in the Supplementary Materials Standard library construction for Illumina Hiseq2000 sequencing platform
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
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Data processing |
For Hi-C, Reads from each end of a DNA fragment were represented in each of the two FASTQ files generated by Illumina paired-end sequencing. For Hi-C, we mapped reads in each FASTQ file back to the genome of Caulobacter CB15N (NA1000) independently by Bowtie version 0.12.7 to preserve the order of reads. All the processing steps afterwards were performed using an in-house Perl scripts. For RNA-seq, the same mapping procedure was used as for Hi-C but coverage was computed using BEDTools instead (Quinlan and Hall, 2010). The two resulting SAM files were then merged together. Unaligned reads were discarded. The Caulobacter CB15N (NA1000) genome was then divided into restriction fragments (700 BglII fragments and 2025 NcoI fragments). Each aligned read was sorted into its corresponding restriction fragment. We inferred that a DNA fragment resulted from non-ligation or self-ligation if reads from both ends were from the same restriction fragment; these reads were discarded. Only DNA fragments for which the reads came from different restriction fragments were retained and used for construction of a Hi-C contact map. The genome of Caulobacter CB15N was then divided into bins of either 20 kb (for BglII Hi-C) or 10 kb (for NcoI Hi-C due to higher cutting frequency of NcoI compared to BglII) and the remaining reads were allocated to their corresponding bin. We then counted the number of fragments having reads with in different bins. A raw Hi-C contact map is the matrix of read counts in which each entry, mij, indicates the number of fragments with ends mapping to bins i and j. The raw Hi-C contact map/matrix is biased due to the uneven distribution of restriction enzyme sites and, to a lesser extent, differences in GC content and the mappability of individual reads. We normalized raw contact maps using an iterative normalization procedure (Imakaev et al., 2012 PMID: 22941365). Essentially, we converted the number of interactions, or read counts, into Hi-C scores by applying the following equation and iteratively repeating it for the resulting contact map after each cycle: mij =mij * (total reads) / (total reads in bin i * total reads in bin j). Ten iterative normalization rounds were performed. Subsequent analysis and visualisation was done using Perl and R scripts. Genome_build: NC_011916.1 Supplementary_files_format_and_content: Files ending with .matrix.txt: tab-delimited text files of the iteratively-corrected Hi-C contact maps/matrices. Files ending with _coverage_output.txt: tab-delimited text file of nucleotide-resolution coverage from RNA-seq data (column 1: genome ID, column 2: nucleotide position, column 3: coverage)
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Submission date |
Oct 26, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Tung Ba Khanh Le |
E-mail(s) |
tung.le@jic.ac.uk
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Phone |
01603450776
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Organization name |
John Innes Centre
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Department |
Department of Molecular Microbiology
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Lab |
www.tunglelab.org
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Street address |
Colney Lane
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City |
Norwich |
State/province |
Norfolk |
ZIP/Postal code |
NR4 7UH |
Country |
United Kingdom |
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Platform ID |
GPL17001 |
Series (1) |
GSE74364 |
Transcription rate and transcript length drive the formation of chromosomal interaction domain |
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Relations |
BioSample |
SAMN04216102 |
SRA |
SRX1384222 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1918963_Laublab_RNAseq_NA1000_PYE_coverage_output.txt.gz |
15.6 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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