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Status |
Public on Feb 02, 2023 |
Title |
E. coli BL21 whole genome, modP1 overexpression |
Sample type |
SRA |
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Source name |
bacterial culture
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Organism |
Escherichia coli |
Characteristics |
strain: BL21 cell type: bacterial culture genotype: modP1 overexpression
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Growth protocol |
A. pleuropneumoniae isolates were grown on BBL™ blood agar base supplemented with thiamine HCl (0.0005%), NADH (0.0025%), oleic acid bovine albumin complex (5%) consisting of 4.75% bovine serum albumin (Fraction V) in normal saline (with the saline containing 5% NaOH and 0.06% oleic acid) and heat inactivated horse serum (1%) (termed BA/SN) or brain heart infusion (BHI, Oxoid) agar containing β-nicotinamide adenine dinucleotide, NAD (0.01%) at 37°C for 24 h. Escherichia coli strains (DH5α and BL21) were grown overnight in lysogeny broth (LB) supplemented with ampicillin at 100 µg/ml or chloramphenicol at 20 µg/ml as required at 37 °C in a shaker at 200 rpm. For mutant selection, A. pleuropneumoniae strains were grown overnight on BHI-NAD plates with 1 µg/ml of chloramphenicol. The Type III methyltransferase genes were cloned into the pET15b vector (Novagen).
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Extracted molecule |
genomic DNA |
Extraction protocol |
Genomic DNA was isolated using the Sigma GenElute kit, or plasmid DNA isolated with the Qiagen miniprep kit. Nanopore sequencing libraries were prepared from 400 ng of DNA per sample with the Rapid DNA Barcoding kit (RBK004) according to manufacturer’s instructions. A 500 ng pool of 6 indexed plasmid libraries was sequenced on 1 PromethION R9.4.1 flow cells (FLO-PRO002) on a PromethION 24 instrument running MinKNOW 22.03.4. For genomic DNA libraries of E. coli and A. pleuropneumoniae, a 500 ng pool of 10 indexed samples was sequenced on 1 PromethION R9.4.1 flow cells (FLO-PRO002) on a PromethION 24 instrument running MinKNOW 22.08.6.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
PromethION |
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Description |
Ecoli_modP1 modP1_difference.RDS
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Data processing |
For plasmid libraries, reads were base called in super-accuracy mode (dna_r9.4.1_450bps_sup_prom) with demultiplexing, using Guppy 6.0.7 and default parameters. Read depth was downsampled to 400x per plasmid with ont_fast5_api v4.0.1 (https://github.com/nanoporetech/ont_fast5_api), first filtering for read quality with Filtong v0.2.1 (https://github.com/rrwick/Filtlong). DNA modification motif discovery and classification was performed with Nanodisco v1.0.3, down-sampling read depth to 400x per plasmid, aligning reads to the pET15b vector containing the relevant mod insert. For genomic DNA libraries of E. coli and A. pleuropneumoniae, reads were basecalled in high-accuracy mode (dna_r9.4.1_450bps_hac_prom) with demultiplexing, using Guppy 6.1.5 and default parameters, and downsampled to a read depth of 400x per sample. DNA modification motif discovery and classification was performed with Nanodisco v1.0.3, aligning reads to the E. coli and A. pleuropneumoniae complete genomes. Assembly: pET15b plasmid (EMD Millipore, catalog number: 69661), E. Coli genome (ASM584v2), A. pleuropneumoniae genome (57675_E01) Supplementary files format and content: RDS objects containing methylation current difference values computed by Nanodisco Library strategy: Transposase (DNA sequencing)
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Submission date |
Jan 30, 2023 |
Last update date |
Feb 02, 2023 |
Contact name |
Matthew Ritchie |
E-mail(s) |
mritchie@wehi.edu.au
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Organization name |
The Walter and Eliza Hall Institute of Medical Research
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Street address |
1G Royal Parade
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City |
Parkville |
State/province |
Victoria |
ZIP/Postal code |
3052 |
Country |
Australia |
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Platform ID |
GPL33073 |
Series (1) |
GSE224057 |
Actinobacillus pleuropneumoniae encodes multiple phase-variable DNA methyltransferases that control distinct phasevarions |
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Relations |
BioSample |
SAMN32961329 |
SRA |
SRX19215786 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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