GSM1937610: 5'-end sequencing after Syn9 infection of WH7803 at 2h TAP treated. [WH7803_inf_III_2h_race_TAP]; Synechococcus sp. WH 7803; RNA-Seq
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.
GEO Accession
GSM1937610