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Sample GSM1078118

Query DataSets for GSM1078118

Status

Public on Apr 01, 2013

Title

Short Exponential Rep2 (37)

Sample type

SRA

Source name

Cultured bacteria mid-exponential phase

Organism

Sinorhizobium meliloti

Characteristics

development stage: mid-exponential phase
strain: Sinorhizobium meliloti 2011
rna fraction: short (< 200 nucleotides)
treatment: Tobacco Acid Pyrophosphatase (TAP)
fragmentation: none
selected insert size: 20-120 nucleotides

Growth protocol

RNAs were extracted from bacteria in liquid cultures in exponential and stationary growth phases and from 10-day-old nodules in which bacteria were differentiated in nitrogen fixing bacteroids

Extracted molecule

total RNA

Extraction protocol

Harvested cultured bacteria were resuspended and incubated in pre-warmed lysis buffer (1.4% SDS, 4 mM EDTA and 0.4 mg/ml proteinase K) for 20 minutes at 65°C. Lysed cells were then incubated at 4°C for 10 minutes in the presence of 1.5 volumes of 5 M NaCl. Following centrifugation, the pellet containing proteins and cell debris was separated from nucleic acids in the supernatant fraction that were subsequently precipitated by the addition of one volume of isopropanol. Nucleic acid-containing pellets were washed with 70% cold ethanol, dried and resuspended in RNAse free water. Total nucleic acids were processed with mirVana isolation kit (Ambion, Invitrogen) using the total RNA isolation procedure which allowed the extraction of both short and long RNAs. For plant material, root nodules were collected and frozen in liquid nitrogen before ground with a mortar and pestle, and then with a Retsch MM300 mill for 1 min. Total RNA was then immediately extracted with the mirVana Isolation Kit using the total RNA isolation procedure. Bacterial or nodule total RNA preparations were then treated with TURBO DNase (Ambion, Life technologies) to remove residual contaminant DNA. Total RNA preparations were depleted of ribosomal RNAs (rRNAs) by an oligocapture strategy derived from the Plant Ribominus kit (Invitrogen), in which oligonucleotide sets specifically designed to target M. truncatula and S.meliloti rRNAs, as well as the highly abundant S. meliloti tRNA_Ala were used. RNAs were separated in two fractions, short (< 200 nt) and long (> 200 nt), using Zymo Research RNA Clean & Concentrator™-5 columns (Proteigene).
Oriented sequencing with a RNA ligation procedure was carried out by Fasteris SA (Geneva, Switzerland) using procedures recommended by Illumina, with adaptors and amplification primers designed by Fasteris, unless specified (see below). For short RNAs, the Small RNA Sequencing Alternative v1.5 Protocol (Illumina) was used, RNAs were first treated with Tobacco Acid Pyrophosphatase to remove triphosphate at 5’ transcript ends, and purified on acrylamide gel before and after the adaptor ligation step. The 3’ adaptor was the Universal miRNA cloning linker (NEB). For long RNAs, a fragmentation step by zinc during 8 min was included, following the Illumina procedure. The size of selected inserts was 20-120 nt for short RNA libraries and 50-120 nt for long RNA libraries from cultured bacteria and 150-250 nt for long RNA libraries from nodules. Libraries were sequenced either in paired-end or single-end.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Data processing

Reads were mapped to the genome using glint (Faraut T. and Courcelle E.; http://lipm-ioinfo.toulouse.inra.fr/download/glint/, unpublished) and parameters were as follow: matches with less than or equal to 2 mismatches, no gap allowed and a minimum length of 18 nt required. We removed all ambiguous matches (reads or read-pairs mapping to two different positions with the same best score) as well as matches of paired-end reads that were separated by more than 1kb.
Genome_build: Sinorhizobium meliloti 2011 submitted to Genbank (pending; GDSub21246)
Supplementary_files_format_and_content: To annotate the Sinorhizobium meliloti genome, we computed the local library specific transcription depth at each position of the sequence (of the chromosome GMI11495-Rm2011G.c and of the megaplasmids GMI11495-Rm2011G.a and GMI11495-Rm2011G.b). The three tabulated files are composed of 20 columns: column 1=position, column2=strand, columns 3-20=local transcription depth for a specific library.

Submission date

Feb 05, 2013

Last update date

May 15, 2019

Contact name

Erika Sallet

E-mail(s)

[email protected]

Organization name

INRA/CNRS

Department

Plant Health and the Environment