title: GSM1151289: Induction 0min Rep1; Escherichia coli str. K-12 substr. MG1655; RNA-Seq
library_construction_protocol: RNA was extracted from monosomes by the hot acid phenol method. RNA dephosphorylated by T4 polynucleotide kinase (M0236, NEB) for 1 h at 37 °C. RNA was loaded onto a 15% Mini-PROTEAN® TBE-Urea Precast Gel (BIO-RAD) and run until the ~30 nt region was resolved.  The region from 28nt to 42nt was excised, based on the mobility of defined RNA oligonucleotides and a 10bp DNA ladder. The gel fraction was eluted overnight in 300 mM NaOAc pH 5.5, 1 mM EDTA, and 0.1U/ul SUPERas In (Ambion #AM2694), followed by ethanol precipitation. PolyA tails were added to the purified RNA fragments by E. coli polyA polymerase (NEB) at 37 °C for 20 min in the buffer provided.  The tailed RNA molecules were reverse transcribed using barcoded primers and SuperScript III (Invitrogen) to generate the first-strand cDNA. After reverse transcription RNA was removed from RNA-DNA duplexes by incubation for 15 min at 98° in 0.1M NaOH, followed by addition of an equal concentration of HCl.  Reverse-transcription products were loaded onto a 10% polyacrylamide TBE-urea gel. The band of the first-strand cDNA synthesis was excised and recovered using DNA gel elution buffer (300mM NaCl, 1mM EDTA). Purified first-strand cDNA was circularized by 50U CircLigase (Epicentre). Circularized ribosomal footprint cDNA was amplified by PCR using the Phusion High-Fidelity enzyme (New England Biolabs) according to the manufacturer’s instructions. The PCR products were separated on a non-denaturing 8% polyacrylamide TBE gel.  Mixed DNA samples from different barcoded samples typically were used for cluster generation. Deep sequencing was performed with the Illumina HiSEQ2000.