title: GSM4873501: E. coli 042 wt (aggR3UTRFRT control); Escherichia coli 042; RNA-Seq
library_construction_protocol: Total RNA was isolated from the cell pellets using the RNAsnap protocol developed by Stead et al. 2012. The total RNA preparations were examined by capillary electrophoresis. From the total RNA samples, ribosomal RNA molecules were depleted using the in-house developed depletion probes. The ribodepleted RNAs were first fragmented using ultrasound (2 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' ends of the RNA molecules. First-strand cDNA synthesis was performed using M-MLV reverse transcriptase and the 3' adapter as primer. The first strand cDNA was purified and the 5' Illumina TruSeq sequencing adapter was ligated to the 3' end of the antisense cDNA. The resulting cDNA was PCR-amplified to about 10-20 ng/μl using a high fidelity DNA polymerase. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis. For Illumina NextSeq sequencing, the cDNAs were pooled in approximately equimolar amounts. The cDNA pool was fractionated in the size range of 200 – 500 bp using a differential clean-up with the Agencourt AMPure kit. An aliquot of the size fractionated cDNA pool was analyzed by capillary electrophoresis. The cDNAs have a size range of 200 - 500 bp. The primers used for PCR amplification were designed for TruSeq sequencing according to the instructions of Illumina. The cDNA pool was paired-end sequenced on an Illumina NextSeq 500 system using 75 bp read length.
attributes.tag: GEO Accession
attributes.value: GSM4873501