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Sample GSM1937622

Query DataSets for GSM1937622

Status

Public on Nov 13, 2015

Title

5'-end sequencing after Syn9 infection of WH8103 at 1h NO TAP. [WH8109_inf_II_1h_race_NOTAP]

Sample type

SRA

Source name

Synechococcus sp. strain WH8109 infected by Syn9

Organism

Synechococcus sp. WH 8109

Characteristics

infection: Infected by Syn9
time: 60min
replicate: 2
tap treated: -

Treatment protocol

Synechococcus cultures were infected at a multiplicity of infection (MOI) of 2-3 approximately 2 h after “lights on” in the morning and incubated under growth conditions. The infection cycle was complete 4 h before darkness. Samples were collected at different time points during the lytic cycle for RNA-sequencing (5 min, 0.5 h, 1 h and 2 h after infection).

Growth protocol

Synechococcus strains were grown in an artificial sea water-based medium (ASW) (Wyman et al., 1985) with modifications as described in Lindell et al. (1998), at 22 °C under cool white light with a 14:10 h light:dark cycle at an intensity of 30 μmol photon·m-2·s-1 during the light period

Extracted molecule

total RNA

Extraction protocol

Synechococcus cells (50-75 ml of each infected or control culture) were collected on 0.4 μm pore-sized polycarbonate filters (GE) by filtration at a vacuum pressure of 10 inch Hg and snap frozen at -80 ºC in 2 ml PGTX buffer (40 ml liquid phenol 95%, 6.9 ml glycerol, 0.1 g 8-hydroxyquinoline, 0.58 g EDTA, 0.8 g sodium acetate, 0.5 g guanidine thiocyanate and 4.6 g guanidine hydrochloride at final pH = 4.2 in a final volume of 100ml) as described in Pinto et al (2009) but without Triton X-100. Each culture was sampled a minute apart such that WH8102-infected cells were sampled 1 and 2 minutes after WH8109- and WH8109-infected cells, respectively. Total RNA was extracted by the PGTX 95 method (Pinto et al. 2009) with some minor modifications. Briefly, the samples were thawed on ice and subjected to a 5-min incubation at 95 °C followed by a further 5-min on ice. The aqueous phase was extracted with an equal volume of chloroform:isoamyl alcohol (24:1, vol/vol). RNA was precipitated with 1 volume of isopropanol and washed in 75% ethanol. Total nucleic acids were quantified based on absorption at 260 nm and RNA integrity was verified by gel electrophoresis. DNA was removed by DNase I digestion using the Turbo DNA-free kit (Ambion). This treatment was repeated to digest residual DNA. RNA samples were then treated with MICROBExpres Bacterial mRNA Purification Kit (Ambion) to remove rRNA. If necessary the rRNA treatment was repeated. Samples were stored at -80°C.
The whole-transcriptome cDNA libraries were prepared using Illumina’s TruSeq RNA Sample Preparation Kit without the polyA isolation step. cDNA libraries for bisulfite strand-specific sequencing were prepared as previously described (Edelheit et al., 2013). 5’-end sequencing libraries were prepared as previously described, with some modifications (Wurtzel et al., 2012a, 2012b): For each sample, 1.5 μg of rRNA-depleted RNA was incubated with or without 5 U of TAP for 2 h at 37°C to generate 5′ monophosphate RNAs (TAP+ and TAP- samples, respectively). The 3′ ends were blocked with NaIO4 (10 mM) for 1.5 h at 4°C in the dark and saturated by adding 1/10 volume of 1 M lysine for 10 min at room temperature. Each reaction was passed through NucAway spin columns (Ambion). Illumina 5′ adapters (in single-strand RNA form), with addition of a unique 4-base index sequence, were ligated to the resultant RNA using 25 units of T4 RNA ligase (Epicentre) in the presence of 40 units of RNase inhibitor (Promega) in a final volume of 50 μL. The reaction was incubated at 37°C for 2 h and precipitated with 1/10 volume of 5 M NH4OAc and 100% ethanol, at 13,000 rpm for 15 min at 4°C. The ethanol was removed after chilling at -80°C for 1 h. The RNA pellet was washed twice with 70% ethanol, resuspended in RNase-free water and washed twice with ultra-pure water on YM-30 microcon columns (Millipore). First strand cDNA was synthesized using random hexamer primers attached to the Illumina 3′ adapter with the SuperScript First Strand Synthesis system (Invitrogen). Adaptors and primer dimers were removed from the resultant cDNA using a PCR purification kit (Qiagen). The cDNA was then PCR amplified using primers matching the 5′ and 3′ Illumina adapters for 20 cycles. Fragments 250-500 bp long were extracted from 3% low melting point agarose gels and quantified with the Bioanalyzer. 5'-end cDNA libraries of 2-10 subsamples were mixed in equal amounts and sent for Illumina sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Synechococcus sp. strain WH8109 infected with theT4-like myovirus Syn9

Data processing

RNA-seq reads were aligned separately for each sample to the reference genomes of the Syn9 cyanophage (Genbank: NC_008296) and the matching host (Genbank: NC_009481, NC_005070 and CP006882 for WH7803, WH8102 and WH8109, respectively), and analyzed according to the library type. Reads that mapped to more than one position were discarded from further analyses.
Reads from the whole-transcriptome libraries were aligned using Novoalign 2.08.01 (Novocraft Technologies Sdn Bhd, http://www.novocraft.com) with the default parameters and [-c 1 -r Random]. reads covering each gene were counted (in-house script), and normalized by gene length and by the number of reads mapped to the RNA component of the RNase P gene (rnpB) from the respective hosts.
Reads from the bisulfite strand-specific libraries were aligned using Novoalign 2.08.01 with the parameters [-b] for the indexing step, and [-r Random] for the alignment. Reads were used to estimate asRNA length, based on the sharp decline in coverage of bisulfite sequencing reads.
Reads from the 5’-end libraries were used for determining TSSs across the phage genome, and were analyzed as previously described (Wurtzel et al., 2012a) with some minor modifications. Briefly, TSS determination relies on the enrichment of TSSs within libraries treated by the Tobacco Acid Pyrophosphatase (TAP), which specifically cleaves primary 5’ triphosphate containing ends, compared to untreated ones, which provides both primary and processed 5’ ends. To differentiate between reads initiating from TSSs versus RNA processing sites, a set of 5’ ends most likely representing real TSSs (positive set) was deﬁned and compared with a group of 5’ ends most likely representing processed sites (negative set). For each time point, the positive set included the 5’ ends located upstream of genes (up to 100 nt) that were reproducible in the phage while infecting all 3 hosts. If multiple genomic positions were found for a gene within this distance, the position with the highest number of supporting reads was selected as the TSS for the positive set. Additional 5’ ends farther than 100nt upstream of genes, for which a sharp increase (>5 fold) in the measured expression produced from the whole-transcript sequencing was observed, were also added to the positive set. The negative set was defined as genomic positions located more than 150 nt inside ORFs. The TAP‏ enrichment threshold for a TSS was calculated for each sample separately based on the distribution of the TAP+/TAP- ratio in the positive and negative sets (Wurtzel et al., 2012a). A genomic position was considered a real TSS at a specific time point, if it was above the TAP+/TAP- threshold in all three hosts or when found in two hosts for multiple time points. Also, positions supported by a sharp increase in coverage were added. The dominant TSS upstream of each gene (up to 400 bp) at each time point was considered to be that gene’s primary TSS. TSSs internal to ORFs were associated with the gene only if they were supported by an increase in coverage, affecting the expression level of that gene. When no TSS was associated with a gene at a certain time point the gene was considered to be part of an operon with the upstream gene under the condition that it was both positioned on the same strand and expressed at the same levels (less than 2 fold change in the median coverage).
Genome_build: Genbank: NC_008296, NC_009481, NC_005070 and CP006882 for Syn9 cyanophage, Synechococcus WH7803, WH8102 and WH8109, respectively.
Supplementary_files_format_and_content: 3 tab-delimited text files, one per host including all time points (controls, ~5 min, 0.5h, 1h, 2h): (A-H) Gene information. Col H is feature - 1/8 for coding/tRNA (I-S) raw reads (T-AD) rnpB normalized reads. Expression of all genes is normalized by the expression of rnpB gene, and by their length. (AE-AH) averages of duplicates. Aabbreviations: controls: c05, c1, c2 7803: a=rep I, b=rep III 8102: a=rep II, b=rep III 8109: a=rep II, b=rep III

Submission date

Nov 11, 2015

Last update date

May 15, 2019

Contact name

Shany Doron

E-mail(s)

shany.doron@weizmann.ac.il

Organization name

Weizmann Institute of Science,

Department

Department of Molecular Genetics