GSM4391887: CFT073 delta Fur RNA Replicate 1; Escherichia coli CFT073; RNA-Seq
RNA was extracted by adding 10 mL of cultures at OD 600nm=0.25  to 1.25 mL Stop Solution (5% water saturated phenol and 95% ethanol).Cells are spun at 9000 rpm for 5 min at 4°C, the liquid is removed and the pellet were resuspended in TE, lysozyme and 10% SDS and heated at 65°C for 2 minutes. Sodium acetate was added and the sample was added to an equal volume of phenol at 65°C and incubated for 6 min with agitation every 40 seconds. Samples were spun at 14,000 rpm at 4°C and the aqueous layer was transferred to equal volume of chloroform. Samples were spunat 14,000 rpm at 4°C and the aqueous layer was transferred to 1/10 volume sodium acetate and 2 volumes ethanol for ethanol precipitation. Dried pellets were resuspended in 200 μL DEPC treated H2O and incubated with DNAse I (2 U) at 37°C for 30 minutes. DNAse I was inactivated  with using equal volumes of phenol and/or chloroform in a phenol extraction, a phenol/chloroform extraction and two chloroform extractions, followed by ethanol precipitation. RNA was resuspended in 50 μL DEPC-treated H2O. Strand–specific RNA-sequencing libraries were prepared using NEBNext Ultra RNA Library Prep Kit (NEB, Ipswich, MA, USA) as per manufacturer's instruction. Briefly, enriched RNAs were fragmented for 15 minutes at 94°C. First and second strands of cDNA were sequentially synthesized, end repaired, adenylated at 3' ends and universal adapters were ligated, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent TapeStation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA) using the adaptor sequences as primers. The sequencing libraries were multiplexed and clustered on one lane of a flow cell. After clustering, the flow cell was loaded on the Illumina HiSeq instrument as per manufacturer's instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq were converted into fastq.gz files and de-multiplexed using Illumina's bcl2fastq.gz 2.17 software. One mismatch was allowed for index sequence identification. Library generation and sequencing was conducted at Genewiz (NJ, USA).