GSM4289286: WT rep2 MLP; Bacteroides thetaiotaomicron; RNA-Seq - SRA

SRX7648628: GSM4289286: WT rep2 MLP; Bacteroides thetaiotaomicron; RNA-Seq
1 ILLUMINA (NextSeq 500) run: 6.9M spots, 522.8M bases, 196.2Mb downloads

Submitted by: NCBI (GEO)

Study: High-resolution view at the coding transcriptome and noncoding RNAs of major gut microbe Bacteroides thetaiotaomicron

PRJNA603791 • SRP245921 • All experiments • All runs

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Gram-negative, obligate anaerobic Bacteroides thetaiotaomicron is emerging as the model organism for gut microbiota research. Colonization of its host niche within the human colon depends to large parts on the extensive metabolic capacity encoded in the Bacteroides genome, but little is known about how its transcription is organized. Here, we transferred differential RNA-seq (dRNA-seq) to B. thetaiotaomicron type strain VPI-5482 to globally map transcription start sites (TSSs). Our screen identified ~4,500 TSSs and we globally determined untranslated regions (UTRs), operon structures, and promoter motifs. In addition, dRNA-seq led to the discovery of ~250 noncoding RNA elements, including 124 novel intergenic small RNAs (sRNAs), several of which we validated experimentally. One of them is the conserved, 145 nt-long sRNA BTnc035, that was highly expressed when B. thetaiotaomicron was grown in the presence of N-acetylglucosamine or glucuronic acid as the sole carbon source. Combining computational predictions with experimental data, we determined the secondary structure of BTnc035 and identified target genes of this sRNA. The emerging model places BTnc035 at the center of a metabolic feedback-loop, wherein sensing of N-acetyl-D-glucosamine and D-glucuronic acid induces BTnc035, which in turn may contribute to a metabolic switch from polysaccharide biosynthesis to break-down. Together, we compiled a single-nucleotide resolution transcriptome map of B. thetaiotaomicron and provide the first global analysis of noncoding RNAs in this important anaerobic model bacterium. To allow easy interrogation of our transcriptome data, we developed Theta-Base – an intuitive online browser that can be freely accessed at https://www.helmholtz-hiri.de/en/bacteroides. Overall design: Samples 1-18: Genome-wide mapping of transcription start sites via differential RNA-seq (dRNA-seq) of Bacteroides thetaiotaomicron VPI-5482 (wild type) grown in TYG to early-logarithmic phase (ELP), mid-logarithmic phase (MLP) and stationary phase (stat). Three biological replicates per strain and condition. Samples 19-24: Comparative expression analysis by RNA-seq of B. thetaiotaomicron wild-type (tdk deletion background), ?BTnc035 (BTnc035 deletion mutant), and BTnc035+ (BTnc035 complementation strain). Two biological replicates per strain.

Sample: WT rep2 MLP

SAMN13941634 • SRS6078247 • All experiments • All runs

Organism: Bacteroides thetaiotaomicron

Library:

Instrument: NextSeq 500

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: SINGLE

Construction protocol: Total RNA was isolated by hot phenol extraction from 4 OD equivalents of culture to which 1.6 mL stop mix (Eriksson et al., 2003) was added (95% v/v ethanol, 5% v/v water saturated phenol, pH >7.0). The cells were lysed by incubation with 600 μL lysozyme (0.5 mg/mL) and 60 μL 10% SDS for 2 min at 64°C, followed by the addition of 66 μL 3 M NaOAc. Phenol extraction (750 μL; Roti-Aqua phenol) was performed at 64°C for 6 min with the subsequent addition of 750 µL of chloroform. RNA was precipitated from the aqueous phase with twice the volume of 30:1 (ethanol : 3 M NaOAc, pH 6.5) mix and incubated at –80°C overnight. After centrifugation, pellets were washed with 75% (vol/vol) ethanol and re-suspended in 50 μL H2O. Contaminating genomic DNA was removed by treating 40 μg total RNA with 5 U of DNase I (Fermentas) and 0.5 μL Superase-In RNase Inhibitor (Ambion) in a 50 μL reaction (1 U/µL, Fermentas). RNA quality was checked using a 2100 Bioanalyzer and the RNA 6000 Nano kit (Agilent Technologies). RNA integrity (RIN) values for all samples were >7. Samples 1-18: Prior to cDNA synthesis, total RNA was fragmented using ultrasound (4 pulses of 30 sec at 4°C) and treated with T4 Polynucleotide Kinase. Subsequently, half the total RNA of each sample was treated with Terminator exonuclease (TEX) to enrich for primary transcripts. RNA samples were then poly (A)-tailed using poly (A) polymerase and 5'-PPP was removed with 5' Polyphosphatase. RNA adaptors were ligated and first-strand cDNA synthesis was carried out using oligo (dT) primers and M-MLV reverse transcriptase. The cDNA was PCR amplified to about 10-20 ng/μL, purified using Agencourt AMPure XP kit and fractionated in a size range of 200-500 bp. Samples 19-24: cDNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep kit for Illumina in accordance with the manufacturers' instructions with modifications: RNA samples were fragmented with Mg2+ at 94˚C for 2.75 min using the NEBNext Magnesium RNA Fragmentation Module followed by RNA purification with the Zymo RNA Clean & Concentrator kit. Fragmented RNA was dephosphorylated at the 3' end, phosphorylated at the 5' end and decapped using 10 U T4-PNK +/- 40 nmol ATP and 5 U RppH, respectively. After each enzymatic treatment, RNA was purified with the Zymo RNA Clean & Concentrator kit. The small RNA fragments were ligated for cDNA synthesis to 3' SR adapter and 5' SR adapter diluted 1:3 with nuclease-free water before use. PCR amplification to add Illumina adaptors and indices to the cDNA was performed for 14 cycles with 1:3 diluted primer. Barcoded DNA libraries were purified using magnetic MagSi-NGSPREP Plus beads at a 1.8 ratio of beads to sample volume. Samples 1-18: dRNA-seq; samples 19-24: RNA-seq

Experiment attributes:

GEO Accession: GSM4289286

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 6.9M spots, 522.8M bases, 196.2Mb

Run	# of Spots	# of Bases	Size	Published
SRR10987167	6,944,594	522.8M	196.2Mb	2020-06-11

ID:: 9979486

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