GSM1151305: WT 10min Rep1; Escherichia coli str. K-12 substr. MG1655;... - SRA

SRX287645: GSM1151305: WT 10min Rep1; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
1 ILLUMINA (Illumina HiSeq 2000) run: 22.8M spots, 1.1G bases, 663Mb downloads

Submitted by: Gene Expression Omnibus (GEO)

Study: High resolution view of bacteriophage lambda gene expression by ribosome profiling

PRJNA205933 • SRP023499 • All experiments • All runs

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Bacteriophage lambda is one of the most extensively studied organisms, and has been a primary model for understanding basic modes of genetic regulation. Here we examine the progress of lambda gene expression during phage development by ribosome profiling, and thereby provide a very high resolution view of lambda gene expression. The known genes are expressed in a predictable fashion, authenticating the analysis. But many previously unappreciated potential open reading frames become apparent in the expression analysis, revealing an unexpected complexity in the pattern of lambda gene function. Overall design: We chose temperature induction of the classic cI857 repressor mutation in a lysogen of E. coli MG1655 in order to synchronize the lytic process, sampling the lysogen and control non-lysogen both before and 2, 5, 10, and 20 minutes after shifting the temperature from 32° to 42°. The last sample time was chosen to be before any significant cell lysis, but during the later stages of lytic gene expression. Total protected nucleotides within open reading frames were summed to determine the density of translation of each reading frame. We take this number to indicate the overall rate of translation, although obviously this assumes that pauses in translation do not excessively affect the overall rate. Since the expression level is not normalized for the copy number of the replicating phage DNA, it thus encompasses both the effect of DNA template availability on mRNA synthesis and the efficiency of utilization of messengers by ribosomes.

Sample: WT 10min Rep1

SAMN02183369 • SRS430304 • All experiments • All runs

Organism: Escherichia coli str. K-12 substr. MG1655

Library:

Instrument: Illumina HiSeq 2000

Strategy: RNA-Seq

Source: TRANSCRIPTOMIC

Selection: cDNA

Layout: SINGLE

Construction protocol: RNA was extracted from monosomes by the hot acid phenol method. RNA dephosphorylated by T4 polynucleotide kinase (M0236, NEB) for 1 h at 37 °C. RNA was loaded onto a 15% Mini-PROTEAN® TBE-Urea Precast Gel (BIO-RAD) and run until the ~30 nt region was resolved. The region from 28nt to 42nt was excised, based on the mobility of defined RNA oligonucleotides and a 10bp DNA ladder. The gel fraction was eluted overnight in 300 mM NaOAc pH 5.5, 1 mM EDTA, and 0.1U/ul SUPERas In (Ambion #AM2694), followed by ethanol precipitation. PolyA tails were added to the purified RNA fragments by E. coli polyA polymerase (NEB) at 37 °C for 20 min in the buffer provided. The tailed RNA molecules were reverse transcribed using barcoded primers and SuperScript III (Invitrogen) to generate the first-strand cDNA. After reverse transcription RNA was removed from RNA-DNA duplexes by incubation for 15 min at 98° in 0.1M NaOH, followed by addition of an equal concentration of HCl. Reverse-transcription products were loaded onto a 10% polyacrylamide TBE-urea gel. The band of the first-strand cDNA synthesis was excised and recovered using DNA gel elution buffer (300mM NaCl, 1mM EDTA). Purified first-strand cDNA was circularized by 50U CircLigase (Epicentre). Circularized ribosomal footprint cDNA was amplified by PCR using the Phusion High-Fidelity enzyme (New England Biolabs) according to the manufacturer’s instructions. The PCR products were separated on a non-denaturing 8% polyacrylamide TBE gel. Mixed DNA samples from different barcoded samples typically were used for cluster generation. Deep sequencing was performed with the Illumina HiSEQ2000.

Experiment attributes:

GEO Accession: GSM1151305

Links:

External link: GEO Sample

NCBI link: NCBI Entrez (gds)

Runs: 1 run, 22.8M spots, 1.1G bases, 663Mb

Run	# of Spots	# of Bases	Size	Published
SRR869832	22,834,526	1.1G	663Mb	2013-07-01

ID:: 407480

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