GSM3334093: Term2; Streptomyces avermitilis; OTHER - SRA

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SRX4556812: GSM3334093: Term2; Streptomyces avermitilis; OTHER
1 ILLUMINA (Illumina HiSeq 2500) run: 12.1M spots, 619.6M bases, 303.6Mb 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: Term2

SAMN09839371 • SRS3673809 • 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. 500~900 ng of the rRNA-depleted RNA was mixed with 1 mL of 150 mM amino-blocked DNA adaptor (5'-p-NNAGATCGGAAGAGCGTCGTGT-3'), 2.5 uL of 10x T4 RNA ligase 1 buffer, 2.5 uL of 10 mM ATP, 2 uL of DMSO, 9.5 uL of 50% PEG8000, and 2.5 uL of T4 RNA ligase 1 (New England BioLabs). The mixture was incubated at 23 oC for 2.5 hours and purified with Agencourt AMPure XP beads (Beckman Coulter) and eluted with 9 uL DEPC-treated water. Then the RNA-adaptor ligates were fragmented using fragmentation buffer (Ambion) by incubating at 72 oC for 90 seconds. After fragmentation, the product was purified with Agencourt AMPure XP beads and eluted with 8 uL DEPC-treated water. The fragmented RNA was reverse transcribed using 1 uL of 10 uM reverse transcription primer (5'-TCTACACTCTTTCCCTACACGACGCTCTTC-3') with SuperScript III Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. After reverse transcription, the cDNA was purified with Agencourt AMPure XP beads and eluted with 5 uL DEPC-treated water. The purified cDNA was subjected to another adaptor ligation as above, with increased incubation time (8 hours) and different amino-blocked adaptor sequence (5'-p-NNAGATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3'). After adaptor ligation, the product was purified using Agencourt AMPure XP beads and indexed by PCR for 10 cycles with Phusion High-Fidelity DNA Polymerase using forward (5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT-3') and reverse (5'-CAAGCAGAAGACGGCATACGAGATNNNNNN (6 nt index) GTGACTGGAGTTCAGAC-3') primers.

Experiment attributes:

GEO Accession: GSM3334093

Links:

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 12.1M spots, 619.6M bases, 303.6Mb

Run	# of Spots	# of Bases	Size	Published
SRR7698517	12,149,121	619.6M	303.6Mb	2020-03-04

ID:: 6165655

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