alias: GSM1676592
title: GSM1676592: TP - SPD 1745 (tprA) and SPD 1746 (phrA) deletion strain, IU6118 rep1; Streptococcus pneumoniae; RNA-Seq
library_construction_protocol: To isolate RNA for RNA-sequencing analysis, starter cultures were used to inoculate 30 ml cultures of BHI broth in 50 mL conical tubes. RNA was extracted from 23 mL of exponentially growing culture (OD620 of ~0.15) using the FastRNA Pro Blue Kit (MP Bio) according to the manufacturer's guidelines. Briefly, cells were isolated by centrifugation at 14,500 x g for 5 minutes at 4°C.  The cells were resuspended in 1 ml RNApro (MP Bio) and processed twice in the Fast Prep Instrument (MP Bio) for 40 seconds at a setting of 6.0. Chloroform and 100% ethanol were used to extract and precipitate the RNA from the resulting lysate and the miRNeasy minikit (Qiagen) and on-column DNase I treatments were used to purify the RNA as above. The amount and purity of all RNA samples isolated were assessed by NanoDrop spectroscopy (Thermo Fisher). RNA integrity was assessed using the Agilent 2100 BioAnalyzer (Aligent Technologies). cDNA libraries were prepared from total RNA by the University of Wisconsin-Madison Biotechnology Center.  The mRNA was enriched from two micrograms total RNA using RiboZeroTM rRNA Removal (Gram-positive bacteria) Kit (EpiCentre Inc.). rRNA-depleted mRNA samples were purified by ethanol precipitation and quantified by fluorometry with the Qubit® RNA assay kit (Invitrogen). Double stranded cDNA synthesis was performed following ScriptSeqTM v2 RNA-Seq Library Preparation guide (EpiCentre Inc.) in accordance with the manufacturer's standard protocol. Thirty nanograms of enriched mRNA were fragmented using divalent cations via incubation for 5 min at 85°C. The first strand of cDNA was synthesized by reverse transcription using random-sequence primers containing a tagging sequence at their 5' ends. Di-tagged cDNA was synthesized by random annealing of a terminal-Tagging Oligo (TTO) to the 3' end of the cDNA for extension of the cDNA by DNA polymerase. Di-tagged cDNA was purified using Agencourt AMPure® XP beads (Beckman Coulter) followed by PCR amplification for 15 cycles using FailsafeTM PCR enzyme and ScriptSeq Index DNA primer set (EpiCentre Inc.). This step generated the second strand of cDNA and completed the addition of Illumina adapter sequences incorporating a user-defined barcode. The amplified libraries were purified using Agencourt AMPure® XP beads. Quality and quantity were assessed using an Agilent DNA 1000 chip (Agilent) and Qubit® dsDNA HS assay kit (Invitrogen), respectively. Libraries were standardized to 2 μM. Cluster generation was performed using standard Cluster kits (v3) and Illumina Cluster Station.
identifiers.namespace: GEO
identifiers.id: GSM1676592