GSM5888799: Grad-seq E. faecalis fraction 11; Enterococcus faecalis V... - SRA

SRX14134044: GSM5888799: Grad-seq E. faecalis fraction 11; Enterococcus faecalis V583; OTHER
1 ILLUMINA (NextSeq 500) run: 17.9M spots, 1.3G bases, 450Mb downloads

External Id: GSM5888799_r1

Submitted by: Jörg Vogel lab, Institute for Molecular Infection Biology, Würzburg University

Study: Enterococcus Grad-seq and KhpB RIP-seq

PRJNA805273 • SRP359294 • All experiments • All runs

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We investigated the RNA-protein interactome of Enterococcus faecalis V583 and Enterococcus faecium Aus0004 by native gradient fractionation of complexes coupled to RNA-sequencing. Whole bacterial cell lysates were analysed by size and density in a glycerol gradient. At native conditions, RNA-protein complexes stay intact and sediment as a whole. Sedimentation profiles of individual RNAs appear correlated in case of interaction in a complex. The profile of KhpB caught our attention and we determined its RNA interactome by immunoprecipitation that suggests a role at the post-transcriptional level, binding notably several tRNAs, sRNAs, and 3'UTRs. Overall design: Cellular lysate was analysed by gradient fractionation into 20 fractions plus the Pellet (P) for E. faecalis and faecium. Subsequently, RNA samples were prepared and sequenced to obtain sedimentation profiles of RNAs. For the RNA-immunoprecipitation of KhpB, we raised two antibodies (Ab1, Ab2) against E. faecalis KhpB, precipitated KhpB from E. faecalis lysate, and sequenced RNA interaction partners, the pre-immune-serum (ctrl1,ctrl2) was used as a negative control.

Sample: Grad-seq E. faecalis fraction 11

SAMN25851575 • SRS11960218 • All experiments • All runs

Organism: Enterococcus faecalis V583

Library:

Name: GSM5888799

Instrument: NextSeq 500

Strategy: OTHER

Source: TRANSCRIPTOMIC

Selection: other

Layout: SINGLE

Construction protocol: Gradient samples were denatured with 1% SDS and RNA was PCI extracted, and ethanol precipitated. 5 µl of the resulting samples were diluted in 45 µl DEPC-treated water. 10 µl were mixedwith 10 µl of a 1:100 diluted ERCC spike-in mix 2 (Thermo Fisher) and subjected to library preparation. In RIP-seq, RNA-protein interaction was denatured by 1% SDS and PCI extraction. RNA was precipitated with ethanol and submitted to library preparation. The RNA samples were fragmented using ultrasound (4 pulses of 30 s at 4°C) followed by 3′-adapter ligation. First strand cDNA synthesis was performed using M-MLV reverse transcriptase. After purification, the 5′-Illumina TruSeq sequencing adapters were ligated to the 3′end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10–20 ng/µl by high-fidelity DNA polymerase and purified with Agencourt AMPure XP kit (Beckman Coulter). cDNA samples were pooled with according to input concentration, a size range of 200–550 bp was eluted from a preparative agarose gel. This size-selected cDNA pool was sequenced on an Illumina NextSeq 500 system using 75 ntsingle-end read length.

Runs: 1 run, 17.9M spots, 1.3G bases, 450Mb

Run	# of Spots	# of Bases	Size	Published
SRR17978247	17,870,962	1.3G	450Mb	2022-11-03

ID:: 19926463

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