PF15 sRNA 25C plate Rep1 - SRA

SRX24651733: PF15 sRNA 25C plate Rep1
1 ILLUMINA (Illumina NovaSeq 6000) run: 100.2M spots, 13G bases, 3.5Gb downloads

Design: Bacterial lawns (plate) for RNA collection were cultured as previously described and grown for 2 days on plates at 25C (or 37C for sequencing). 37C liquid cultures for sequencing were pelleted for collection. Bacterial lawns were collected from the surface of NGM plates using a cell scraper. In brief, 1 ml of M9 buffer was applied to the surface of the bacterial lawn, and the bacterial suspension following scraping was transferred to a 15-ml conical tube. PF15 from 10 plates or OP50 from 15 plates was pooled in each tube and pelleted at 5,000g for 10 min at 4C. The supernatant was discarded, and the pellet was resuspended in 1 ml of Trizol LS for every 100 l of bacterial pellet recovered. The pellet was resuspended by vortexing and subsequently frozen at 80C until RNA isolation. To isolate RNA from bacterial pellets, Trizol lysates were incubated at 65C for 10 min with occasional vortexing. Debris was pelleted at 7,000g for 5 min at 4C. The supernatant was transferred to new tubes containing 1/5 the volume of chloroform. Samples were mixed thoroughly by inverting and centrifuged at 12,000g for 10 min at 4C. The aqueous phase was used at input for RNA purification using the mirVana miRNA isolation kit according to the manufacturers instructions for small RNA (<200 nt) isolation. Purified RNA was used immediately for sRNA training, or for sequencing frozen at 80C until further use.-Sample treated with RNA 5' pyrophosphohydrolase (RppH, NEB) at 37 deg C 30 min-converted to Illumina sequencing libraries using the PrepX RNA-seq library preparation protocol on the automated Apollo 324 NGS Library Prep System (Takara Bio)- libraries were examined on Bioanalyzer DNA High Sensitivity chips (Agilent) for size distribution, quantified by Qubit fluorometer (Invitrogen), and then pooled at equal molar amount and sequenced on Illumina NovaSeq 6000 S Prime flowcell

Submitted by: Princeton University

Study: Pseudomonas fluorescens strain:PF15 Genome sequencing and assembly

PRJNA1114673 • SRP509220 • All experiments • All runs

show Abstracthide Abstract

C. elegans are exposed to a variety of pathogenic and non-pathogenic bacteria species in their natural environment. Correspondingly, C. elegans has evolved an ability to discern between nutritive and infectious bacterial food sources. Here we show that C. elegans can learn to avoid the pathogenic bacteria Pseudomonas fluorescens 15 (PF15), and that this learned avoidance behavior is passed on to progeny for four generations, as we previously demonstrated for Pseudomonas aeruginosa (PA14) and Pseudomonas vranovensis, using similar mechanisms, including the involvement of both the TGF-Beta ligand DAF-7 and Cer1 retrotransposon-encoded virus-like particles. PF15 small RNAs are both necessary and sufficient to induce this transgenerational avoidance behavior. Unlike PA14 or P. vranovensis, PF15 does not use P11, Pv1, or a small RNA with maco-1 homology for this avoidance; instead, an unrelated PF15 small RNA, Pfs1, that targets the C. elegans vab-1 Ephrin receptor gene is necessary and sufficient for learned avoidance, suggesting the evolution of yet another bacterial sRNA/C. elegans gene target pair involved in transgenerational inheritance of pathogen avoidance. As VAB-2 Ephrin receptor ligand and MACO-1 knockdown also induce PF15 avoidance, we have begun to understand the genetic pathway involved in small RNA targeted pathogenic avoidance. Moreover, these data show that axon guidance pathway genes (VAB-1 and VAB-2) have previously unknown adult roles in regulating neuronal function. C. elegans may have evolved multiple bacterial specificity-encoded small RNA-dependent mechanisms to avoid different pathogenic bacteria species, thereby providing progeny with a survival advantage in a dynamic environment.

Sample: PF15 sRNA 25C plate Rep1

SAMN41491976 • SRS21387434 • All experiments • All runs

Organism: Pseudomonas fluorescens

Library:

Name: PF15 sRNA 25C plate Rep1

Instrument: Illumina NovaSeq 6000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: PAIRED

Runs: 1 run, 100.2M spots, 13G bases, 3.5Gb

Run	# of Spots	# of Bases	Size	Published
SRR29128101	100,189,003	13G	3.5Gb	2024-05-22

ID:: 32970613

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