GSM3334091: TSS_TAP_untreated2; Streptomyces avermitilis; OTHER - SRA

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SRX4556810: GSM3334091: TSS_TAP_untreated2; Streptomyces avermitilis; OTHER
1 ILLUMINA (Illumina HiSeq 2500) run: 7M spots, 707.9M bases, 367.8Mb downloads

Submitted by: NCBI (GEO)

Study: Transcriptome and translatome of Streptomyces avermitilis MA-4680

PRJNA486172 • SRP158023 • All experiments • All runs

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The gram-positive bacterium, Streptomyces avermitilis holds industrial importance, which produces widely used anthelmintic agent, avermectin. Furthermore, S. avermitilis is generally considered as a prominent heterologous gene expression host for diverse secondary metabolites biosynthesis. However, despite of its industrial importance, it largely remains unknown how its genome is organized and regulated for timely gene expression. Here, we determined 1,601 transcription units (TU) encoded in its genome using the integrated analysis of high-throughput sequencing data including dRNA-Seq, Term-Seq, RNA-Seq, and Ribo-Seq. In addition to TU cataloguing, these information-rich results also revealed the presence of diverse regulatory elements for the transcriptional and translational control of individual TU, such as promoters, 5¢-UTRs, terminators, 3¢-UTRs, and riboswitches. The conserved promoter sequences for transcription initiation were identified from 2,361 transcription start sites as 5¢-TANNNT and 5¢-TGAC for -10 and -35 elements, respectively. Interestingly, the -35 element and spacer length between them were critical for transcriptional regulation of functionally distinct genes. Total 2,017 transcription termination sites were detected from Term-Seq analysis, revealing that stem structure formation is a prerequisite for transcription termination and that Rho-independent termination prevails in S. avermitilis. Lastly, the TU architecture suggests the presence of novel small RNAs and cis-regulatory elements in the genome. Our findings will serve as invaluable resources for comprehensive understanding on regulatory features of S. avermitilis. Moreover, it is anticipated the elevation of its potential as the heterologous expression host for diverse secondary metabolite biosynthesis. Overall design: Profiles of 5' termini of primary transcripts, 3' termini of whole transcripts, whole transcripts and ribosome protected RNA fragements of Streptomyces avermitilis were generated by deep sequencing using Illumina Hi-Seq 2500

Sample: TSS_TAP_untreated2

SAMN09839373 • SRS3673807 • All experiments • All runs

Organism: Streptomyces avermitilis MA-4680 = NBRC 14893

Library:

Instrument: Illumina HiSeq 2500

Strategy: OTHER

Source: TRANSCRIPTOMIC

Selection: other

Layout: SINGLE

Construction protocol: Harvested cells were washed with polysome buffer (20 mM Tris-HCl pH 7.5, 140 mM NaCl, 5 mM MgCl2), and then resuspended with lysis buffer (0.3 M sodium acetate pH 5.2, 10 mM EDTA, 1% Triton X-100). The cell suspension was then frozen with liquid nitrogen, and lysed by grinding using mortar and pestle. The cell lysate was centrifuged at 4 oC for 10 minutes at 16,000 g and the supernatant was stored at -80 oC until used for RNA extraction. RNA was extracted by mixing with equal volume of phenol:chloroform:isoamyl alcohol = 25:24:1 solution. The mixture was then centrifuged and upper aqueous phase was recovered. The RNA was purified with ethanol precipitation. The purified RNA was treated with DNase I (New England Biolabs) to remove any genomic DNA contamination. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit Bacteria (Epicentre) according to the manufacturer's instructions. About 700 ng of rRNA-depleted RNA was incubated in 1x RNA 5' polyphosphatase (TAP) (Epicentre) reaction buffer and 1 U of SUPERase-In (Invitrogen) at 37 oC for 1 hour without TAP. After ethanol precipitation, 5 pmol of 5' RNA adaptor (5'-ACACUCUUUCCCUACACGACGCUCUUCCGAUCU-3') was ligated to the purified RNA with T4 RNA ligase (Thermo) in 1x RNA ligase buffer and 0.1 mg/mL BSA by incubating at 37 oC for 90 minutes. The adaptor-ligated RNA was then purified using Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer's instructions. The purified product was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) and purified using Agencourt AMPure XP beads. The purified cDNA was amplified and indexed using Phusion High-Fidelity DNA Polymerase (Thermo) for the Illumina sequencing. The amplification step was monitored on a CFX96 Real-Time PCR Detection System (Bio-Rad) to be stopped before the PCR reaction was fully saturated. Finally, the amplified library was purified using Agencourt AMPure XP beads, and the concentration of the library was measured with Qubit 2.0 fluorometer (Invitrogen). The size distribution of the library was checked with gel electrophoresis on 2% agarose gel.

Experiment attributes:

GEO Accession: GSM3334091

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 7M spots, 707.9M bases, 367.8Mb

Run	# of Spots	# of Bases	Size	Published
SRR7698515	7,009,201	707.9M	367.8Mb	2020-03-04

ID:: 6165653

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