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| Status |
Public on May 21, 2024 |
| Title |
HrAgo1-associated small RNAs |
| Sample type |
SRA |
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| Source name |
BL21-Gold (DE3)
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| Organism |
Escherichia coli |
| Characteristics |
cell line: BL21-Gold (DE3)
treatment: No treatment
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| Treatment protocol |
The pelleted cells were lysed by sonication (Daxluot Multichannel sonicator with 6mm tip, 5s ON/8 s OFF for 1 hour under constant cooling circulation) in Lysis Buffer (500mM NaCl, 5 mM Imidazole, 50 mM Tris-HCl pH 8) supplemented with 1mM PMSF and a protease inhibitor cocktail (Roche). TCEP was added immediately after sonication to a concentration of 1mM. After centrifugation at 6000 x g for 30 minutes at 4C, the cell-free extract was loaded on 5 ml HisTrap HP column (Cytiva Life Sciences) which was subsequently washed with Washing Buffer I (500 mM NaCl, 20 mM Imidazole, 1mM TCEP, 50 mM Tris-HCl pH 8). Bound protein was eluted with 15 ml Elution Buffer (500 M NaCl, 500 mM Imidazole, 5% glycerol, 50 mM Tri-HCl pH 8). The sample was concentrated to a volume of 2 ml using 30kDa Amicon ultracentrifugal filter. Imidazole-free buffer (500 M NaCl, 5% glycerol, 50 mM Tri-HCl pH 8) was added, well-mixed and re-concentrated three times to replace the original buffer. The final round of centrifugation was done to result in approximately 1ml solution, moved to a fresh 1.5 ml tube and was centrifuged for 30 min at 16,000 x g at 4C to remove aggregates. The supernatant was loaded on a Superdex 200 Increase 10/300 GL column (Cytiva) for size exclusion. The peak fractions were analyzed by SDS-PAGE and fractions containing HrAgo1 were combined and concentrated, aliquoted and flash frozen in liquid nitrogen before storage at -80C until further use.
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| Growth protocol |
E.coli culture was inoculated into Terrific medium supplemented with 50 μg/ml Kanamycin and was shaken at 180 rpm at 37°C in an incubator until an OD600 nm of 0.4. IPTG was added to reach a final concentration of 0.1 mM, and the culture was moved to 24 °C and kept shaking for 8 hours. Cells were harvested by centrifugation at 4,250 x g at 4°C for 20 minutes
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| Extracted molecule |
other |
| Extraction protocol |
To extract nucleic acids co-purified with HrAgo1, 2 nmoles of purified protein was incubated with 250 µg/ml proteinaseK (Thermo Scientific) for 4h at 65C. Next, phenol:chloroform:IAA 25:24:1 pH 7.9 (Invitrogen) was added in a 1:1 ratio. The sample was vortexed and centrifuged at 16000 x g in a table top centrifuge for 10 min. The upper layer containing the nucleic acids was transferred to a clean tube and the nucleic acids were precipitated through ethanol precipitation. To this end, 99% cold ethanol and 3 M sodium acetate pH 5.2 were added to the sample in a 2:1 and 1:9 ratio, respectively. The sample was incubated overnight at -80°C, after which it was centrifuged at 16000 x g in a table top centrifuge for 1 h. The pellet was washed with 70% ethanol and subsequently dissolved in nuclease-free water.
The small RNAseq library was constructed by GenomeScan (Leiden, NL) using standard small RNA library preparation protocols
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| Library strategy |
ncRNA-Seq |
| Library source |
other |
| Library selection |
size fractionation |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
HrAgo1_sRNA
Protein-nucleic acid complexes purified from E. coli, nucleic acids extracted, small RNA sequencing protocol
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| Data processing |
Paired-end small DNA reads were merged and adapter sequences were trimmed using BBmap (Bbtools v38.90; https://pubmed.ncbi.nlm.nih.gov/29073143/). Max length was set at 35 nucleotides
Reads were aligned against the genome of E. coli BL21(DE3) (CP053602.1) and protein expression plasmids (pFWC01) using HISAT2 v2.1.0 (https://pubmed.ncbi.nlm.nih.gov/25751142/)
Mapped reads were filtered using Samtools and analyzed using FastQC to determine read length and to investigate nucleotide bias
Aligned reads were analyzed using FeatureCounts to determine mapping frequence on genomic and plasmid features.
Assembly: Reads were aligned against the genome of E. coli BL21(DE3) (CP053602.1) and protein expression plasmid (pFWC01)
Supplementary files format and content: Analysis to which genome/plasmid reads map and fastqc read length and nucleotide bias analysis (sRNA_analyses.xlsx)
Supplementary files format and content: FeatureCounts analyses for each genome/plasmid present (FeatureCounts_results.xlsx)
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| Submission date |
May 15, 2024 |
| Last update date |
May 21, 2024 |
| Contact name |
Daan C. Swarts |
| E-mail(s) |
daan.swarts@wur.nl
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| Organization name |
Wageningen University
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| Department |
Laboratory of Biochemistry
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| Lab |
Swarts lab
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| Street address |
Stippeneng 4
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| City |
Wageningen |
| ZIP/Postal code |
6708WE |
| Country |
Netherlands |
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| Platform ID |
GPL25368 |
| Series (1) |
| GSE267550 |
RNA-guided RNA silencing by an 1 Asgard archaeal Argonaute |
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| Relations |
| BioSample |
SAMN41405103 |
| SRA |
SRX24564359 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8268680_FeatureCounts_results.xlsx |
291.2 Kb |
(ftp)(http) |
XLSX |
| GSM8268680_sRNA_analyses.xlsx |
38.8 Kb |
(ftp)(http) |
XLSX |
SRA Run Selector |
| Raw data are available in SRA |
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