title: NextSeq 500 sequencing; Transcription start site identification in wild type Escherichia coli
design: Transcription start site identification in wild type Escherichia coli
library_construction_protocol: Duplicate cultures of  E. coli MG1655 were grown until mid-exponential phase in LB media with shaking at 37⁰C. Cells were harvested and flash frozen in liquid nitrogen.  E. coli strain MG1655 was grown until mid-exponential phase in LB media with shaking at 37⁰C Cells were lysed using the RNAsnap protocol (Stead et al.: RNAsnap™: a rapid, quantitative and inexpensive, method for isolating total RNA from bacteria. Nucleic Acids Res. 2012 Nov; 40(20): e156). Total RNA was then purified using the Qiagen Mini RNeasy kit following manufacturers instructions The RNA samples were first fragmented using ultrasound (4 pulses of 30 s each at 4°C). Then, an oligonucleotide adapter was ligated to the 3' end 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 (16 cycles). The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and was analyzed by capillary electrophoresis.
attributes.tag: Experimental Factor: replicate
attributes.value: a