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| Status |
Public on Nov 16, 2016 |
| Title |
CuL-sRNA |
| Sample type |
SRA |
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| Source name |
WT
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| Organism |
Paracoccus denitrificans |
| Characteristics |
genotype/variation: WT
treatment: CuL ANEROBIC
treatment: HIGH N2O ANEROBIC
growth time point (hours from innuculation): 16 h
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| Growth protocol |
P. denitrificans was grown in a defined minimal medium which contained 29 mmol/L Na2HPO4,11 mmol/L KH2PO4,10mmol/L NH4Cl, 0.4 mmol/L MgSO4,and supplemented with 30 mmol/L sodium succinate, 20 mmol/L NaNO3,and 2 mL/L Vishniac and Santer trace elements solution [130 mmol/L EDTA,7.64 mmol/L ZnSO4, 25 mmol/L MnCl2, 18.5 mmol/L FeSO4,0.89 mmol/L (NH4)6Mo7O24, 6.4 mmol/L CuSO4,6.72mmol/LCoCl2, 37.4 mmol/L CaCl] [8]. For high N2O emitting culture conditions, CuSO4 was omitted from the trace elements solution as in Sullivan et al., 2013. Anaerobic batch cultures (200 mL) inoculated with 1% (v/v) of stationary phase cells that had been pre-grown in minimal media. Vessels used were 250 mL Duran bottles with screw-cap lids and gas-tight silicone septa. Cultures were sparged with N2 for 10 minutes to impose an anaerobic environment and incubated statically at 30 oC. Aerobic conditions were created by using 50 ml of media in a 250 ml flask and shaking at 200 rpm at 30 oC
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, 30 mL of mid exponential phase cells (OD600 ≈ 0.4) was added to 12 mL of ice-cold 95% ethanol/5% phenol (pH= 4.3) (v/v) solution, and incubated on ice for 30 min to stabilize RNA and prevent degradation. RNA was isolated, using the Trizol method according to the protocol described in [9]. Trace DNA contamination was removed using Turbo DNA-free DNase (Ambion) and this was confirmed by PCR amplification of RNA samples using MyFi DNA polymerase (Bioline) according to the manufacturer’s instructions. RNA was quantified spectrophotometrically using a Nanodrop 2000 (Thermo Scientific), and integrity of RNA samples was analyzed using an Experion Automated Electrophoresis platform (Bio-Rad) using RNA StdSens chips (Bio-Rad) according to the manufacturer’s instructions.
Library preparation and sequencing were performed by Vertis Biotechnology AG, Germany. Briefly, the total RNA samples were split into two, one was enriched for the small RNA fractions <200 nt (s) specifically using the RNeasy MinElute Cleanup Kit (Qiagen). Ribosomal RNA molecules were depleted from both samples using the Ribo-Zero rRNA Removal Kit for bacteria (Epicentre). The rRNA depleted RNA fractions were first treated with Tobacco Acid Pyrophosphatase (TAP, Epicentre). Afterwards, oligonucleotide adapters were ligated to the 5' and 3' ends of the RNA samples. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3’adapter as primer. The resulting cDNAs were amplified by PCR using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. The cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 bp read length.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Alignment of reads to reference genome (NC_018750.1) using bowtie2 (bowtie2-2.2.3) options [bowtie2 -x reference_genome -S output.sam -U read_file]
Aligned sam file was split into strand specific files using the below script and adding a suitable sam header: use strict; my $file=$ARGV[0]; open (INFIL, "<$file"); open (POS, ">$file.pos.sam"); open (NEG, ">$file.neg.sam"); while (<INFIL>){ chomp; my @ar = split (/\t/, $_); if ($ar[0] =~ m/^@/ or $ar[1] == 163){ print NEG "$_\n"; } if ($ar[0] =~ m/^@/ or $ar[1] == 99){ print POS "$_\n"; } }
conversion to .bam format and sorting using samtools (version 1.2). Commands: samtools view -bS [input.sam] > [output.bam]. samtools [input.bam] [output.sortedbam]
Conversion of sorted bam file to a bedgraph format using bedtools. Command: genomeCoverageBed -ibam sorted.bam -g reference_genome.fna -bga file.bedgraph
production of a wig file suitable for visualisation in IGB using personal unpublished script file contains header (track type=wiggle_0 name=WT_16h graphType=points visibility=full color=168,130,88 fixedStep chrom=Sven start=1 step=1 span=1) with each subsequent line corresponding to the number of times a single base was sequenced with each base represented by a seperate line.
Additional visual inspection was carried out using the wig files and IGB to validate target peaks.
Genome_build: CP000489, CP000490, CP000491
Supplementary_files_format_and_content: wig files were generated using the below perl (v5.8.8) script: =for comment --------------------------------------------------------------------------------------------- this is an example of a bedfile format output from the tools "genomeCoverageBed" in bedtools- --------------------------------------------------------------------------------------------- -some weird reference base base postion position frequency from to i|408675720|ref|NC_018750.1| 0 1 7 gi|408675720|ref|NC_018750.1| 1 2 11 gi|408675720|ref|NC_018750.1| 2 3 12 gi|408675720|ref|NC_018750.1| 3 5 13 gi|408675720|ref|NC_018750.1| 5 7 14 gi|408675720|ref|NC_018750.1| 7 13 15 gi|408675720|ref|NC_018750.1| 13 14 16 gi|408675720|ref|NC_018750.1| 14 15 18 =cut use strict; use warnings; my $file = <STDIN>; my @array; my $from; my $to; my $cc=1; open (BED, "$file"); while (<BED>){ chomp $_; @array = split (/\t/, $_); $from = $array[1]; $to = $array[2]; my $freq= $array[3]; my $count = 0; my $dif = $to-$from; while ($dif-$count>0){ $count++; my $tt = $from + $count; print "$freq\n"; } } close (BED); exit; )
Supplementary_files_format_and_content: 24.1_cuL-sRNA_491.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuL for cp000491
Supplementary_files_format_and_content: 24.2_cuL-sRNA_491.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuL for cp000491
Supplementary_files_format_and_content: 23.1_cuL-sRNA_489 wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuL for cp000489
Supplementary_files_format_and_content: 23.2_cuL-sRNA_489.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuL for cp000489
Supplementary_files_format_and_content: 22.1_cuL-sRNA_490.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuL for cp000490
Supplementary_files_format_and_content: 22.2_cuL-sRNA_490.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuL for cp000490
Supplementary_files_format_and_content: 15.1_cuH-sRNA_491.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuHfor cp000491
Supplementary_files_format_and_content: 15.2_cuH-sRNA_491.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuH for cp000491
Supplementary_files_format_and_content: 13.1_cuH-sRNA_489.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuHfor cp000489
Supplementary_files_format_and_content: 13.2_cuH-sRNA_489.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuH for cp000489
Supplementary_files_format_and_content: 14.1_cuH-sRNA_490.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuHfor cp000490
Supplementary_files_format_and_content: 14.2_cuH-sRNA_490.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuH for cp000490
Supplementary_files_format_and_content: 6.1_cuH-o2-sRNA_491.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuH o2 for cp000491
Supplementary_files_format_and_content: 6.2_cuH-o2-sRNA_491.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuH o2 for cp000491
Supplementary_files_format_and_content: 5.1_cuH-o2-sRNA_490.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuH o2 for cp000490
Supplementary_files_format_and_content: 5.2_cuH-o2-sRNA_490.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuH o2 for cp000490
Supplementary_files_format_and_content: 4.1_cuH-o2-sRNA_489.wig: wig - a file containg the frequency each base was sequenced for the positive strand for CuH o2 for cp000489
Supplementary_files_format_and_content: 4.2_cuH-o2-sRNA_489.wig: wig - a file containg the frequency each base was sequenced for the negative strand for CuH o2 for cp000489
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| Submission date |
Aug 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hannah Gaimster |
| E-mail(s) |
h.gaimster@uea.ac.uk
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| Organization name |
UEA
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| Street address |
Norwich Research Park
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| City |
Norwich |
| ZIP/Postal code |
NR4 7TJ |
| Country |
United Kingdom |
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| Platform ID |
GPL22301 |
| Series (1) |
| GSE85362 |
RNA-seq data of Paracoccus denitrificans grown under different N2O levels |
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| Relations |
| BioSample |
SAMN05544939 |
| SRA |
SRX2009628 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2265843_13.1_cuh-sRNA_489.wig.gz |
769.0 Kb |
(ftp)(http) |
WIG |
| GSM2265843_14.2_cuh-sRNA_490.wig.gz |
409.9 Kb |
(ftp)(http) |
WIG |
| GSM2265843_22.2_cuL-sRNA_489.wig.gz |
519.8 Kb |
(ftp)(http) |
WIG |
| GSM2265843_23.1_cuL-sRNA_490.wig.gz |
276.8 Kb |
(ftp)(http) |
WIG |
| GSM2265843_4.2_cuh-O2-sRNA_489.wig.gz |
611.9 Kb |
(ftp)(http) |
WIG |
| GSM2265843_5.1_cuh-O2-sRNA_490.wig.gz |
326.2 Kb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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