title: GSM2042625: IU8553_RNA-seq rep 3; Streptococcus pneumoniae; RNA-Seq
library_construction_protocol: RNA was prepared from cultures of strains IU6741 (∆pbp1a), IU8553 (∆pbp1a mltG(∆488bp)), IU8567 (∆pbp1a mltG(∆488bp) ∆pbp2b) and IU1824 (pbp1a+ mltG+ pbp2b+ parent). RNA was extracted from 23 ml of exponentially growing culture (OD620 ≈ 0.15) using a FastRNA Pro Blue Kit (MP Bio) according to the manufacturer's guidelines. Briefly, cells were collected by centrifugation at 14, 500 xg for 5 min at 4°C. Cells were resuspended in 1 mL RNApro (MP Bio) and processed twice in a Fast-Prep 24 homogenizer (MP Bio) for 40 s at setting 6.0. Chloroform and 100% ethanol were used to extract and precipitate the RNA from the resulting lysate, and the miRNeasy minikit (Qiagen) with on-column DNase I treatments was used to purify RNA. cDNA libraries were prepared from total RNA by the Center for Genomics and Bioinformatics at Indiana University in Bloomington, Indiana. The mRNA was enriched from 1 μg of total RNA using RiboZero™ rRNA Removal Kit (Bacteria) (EpiCentre, Inc.). rRNA-depleted mRNA samples were purified using RNeasy Minelute Cleanup Kit (Qiagen). Double-stranded cDNA synthesis was performed following ScriptSeq™ Complete Kit (Bacteria) Preparation guide (EpiCentre, Inc.) in accordance with the manufacturer's standard protocol. Entire resultant mRNA sample was 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 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 Failsafe™ PCR enzyme and ScriptSeq™ Index PCR 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 D1000 TapeStation (Agilent Technologies) and Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies), respectively. Sequencing was performed at the Greehey Children’s Cancer Research Institute Genome Sequencing Facility at the University of Texas Health Science Center at San Antonio, Texas. Cluster generation was performed using TruSeq PE Cluster Kit v3-cBot-HS (Illumina). Single-end, 50 bp sequencing was performed using TruSeq SBS Kit v3-HS (Illumina) on a HiSeq 2000 sequencer (Illumina). Image analysis, base calling, sequencing reads de-multiplexing were followed by default Illumina procedure.
attributes.tag: GEO Accession
attributes.value: GSM2042625