title: GSM2190872: 17.4A; Escherichia coli; RNA-Seq
library_construction_protocol: Four ml of the bacterial suspension in RNALater II and Medium E growth medium were spun down at 200g for 5 min. The supernatant was discarded and the pellet were resuspended in 200ul PBS and mixed by pipetting. 100ul aliquots were taken out for DNA and RNA extraction from the same sample. RNA extraction was done using Qiagen RNeasy mini kit (Qiagen, Hilden, Germany) with on-column DNase digestion. Final elution was made in 30 μL dH2O. The concentration and integrity of the total RNA was estimated by Quant-iT™ RiboGreen® RNA Assay Kit  (ThermoFisher Scientific, Waltham, MA), and Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA), respectively. Ribosomal RNA (rRNA) was removed using Ribo-Zero™ Gold (Yeast) kit (Epicenter, Madison, WI) using manufacturer's recommended protocol. Immediately after the rRNA removal the RNA was fragmented and primed for the first strand synthesis using the NEBnext First Strand synthesis module (New England BioLabs Inc., Ipswich, MA). Directional second strand synthesis was performed using NEBNExt Ultra Directional second strand synthesis kit. Following this the samples were taken into standard library preparation protocol using NEBNext® DNA Library Prep Master Mix Set for Illumina® with slight modifications. Briefly, end-repair was done followed by polyA addition and adapter ligation. Post-ligated materials were individually barcoded with primers and amplified through 12 cycles of PCR. Final library quantity was assessed by Quant-iT™ PicoGreen® dsDNA Assay Kit and the library quality was estimated on LabChip® GX (Caliper, PerkinElmer, Waltham, MA). Accurate quantification of the final libraries for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification kit (Kapa Biosystems, Inc., Woburn, MA). Each library was diluted to a final concentration of 12.5 nM and pooled equimolar prior to clustering. Paired-End (PE) sequencing was performed on an Illumina HiSeq2500 sequencer (Illumina, Inc.).
attributes.tag: GEO Accession
attributes.value: GSM2190872