title: GSM3019241: IS_3: IS-knockdown sample 3; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
library_construction_protocol: Total RNA was prepared using the E.Z.N.A. Bacteria RNA Kit (Omega bio-tek, Norcross, GA, USA) following instructions of the manufacturers. Library construction was performed using TrueSeq RNA Library Preparation Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer's instructions with slight modifications. Briefly, RNA quality and quantity measurements were performed using RNA ScreenTape and Reagents on TapeStation (all from Agilent, Santa Clara, CA, USA) and Qubit (Thermo Fisher Scientific, Waltham, MA, USA); only high quality (RIN >8.0) total RNA samples were processed. Next, 1 µg of RNA was DNaseI (Thermo Fisher Scientific, Waltham, MA, USA) treated, the ribosomal RNA depleted using RiboZero Magnetic Kit for Gram-negative bacteria (Epicentre, Madison, WI, USA) and the leftover was ethanol precipitated. The success of rRNA removal was determined by measurement on TapeStation using high-sense RNA ScreenTape and Reagents (all from Agilent, Santa Clara, CA, USA). Next, RNA was purified and fragmented; first strand cDNA synthesis was performed using SuperScript II (Thermo Fisher Scientific, Waltham, MA, USA) followed by second strand cDNA synthesis, end repair, 3'-end adenylation, adapter ligation and PCR amplification. All of the purification steps were performed using AmPureXP Beads (Beckman Coulter, Indianapolis, IN, USA). Final libraries were quality checked using D1000 ScreenTape and Reagents on TapeStation (all from Agilent, Santa Clara, CA, USA). Concentration of each library was determined using the KAPA Library Quantification Kit for Illumina (KAPA Biosystems, Wilmington, MA, USA).
attributes.tag: GEO Accession
attributes.value: GSM3019241