title: GSM2794765: pACYC177 plasmid / fast Luciferase rep 3; Escherichia coli str. K-12 substr. MG1655; OTHER
library_construction_protocol: Once the cultures reached an OD600 of 0.40 - 0.65 (measured using a Shimadzu UV-1800 spectrophotometer; 1 OD600 = 6 x 108 cells / mL) they were filtered through a pre-warmed 0.22 μm nitrocellulose filter paper and 90 mm glass filtration system (Kontes) with vacuum (Whatman cat. no. 7182-009); the filtration took about 1 min. The apparatus was rapidly disassembled, and the cells were immediately scraped from the filter paper, using a pre-warmed scoopula, and submerged into a 50 ml Falcon tube (Corning cat. no. 352070) filled with liquid nitrogen (LN2). The cells were then transferred to a mortar immersed in, and containing, LN2 and 600 μl of cell lysis buffer (10 mM MgCl2, 100 mM NH4Cl, 20 mM Tris-HCl pH 8.0, 0.1% Nonidet P40, 0.4% Triton X-100, 500 μg/ml chloramphenicol) were dripped in. The cells were pulverized with the chilled mortar and pestle, whereupon the LN2 was allowed to boil off. The remaining powder was scraped with a chilled spatula into a chilled 1.5 ml Eppendorf tube and stored at -80°C overnight. Cell lysates were centrifuged 15 min at 14,000 RPM and 4°C, and the clarified supernatant was transferred to a 1.5 ml Eppendorf tube. Between 250 μl and 500 μl of supernatant were then treated with 2.0 μl of micrococcal nuclease (Life Technologies cat. no. EN0181) and 5 mM CaCl2 and incubated 1 h while gently rotating at room temperature. The reactions were quenched using 6 mM EGTA and then transferred to 3.5 ml thickwall polycarbonate ultracentrifuge tubes (Beckman Coulter cat. no. 349622). The samples were underlaid with 2.0 ml of 1 M sucrose and centrifuged 6.5 h on a Beckman XL-80K ultracentrifuge at 55,000 RPM and 4°C in a SW55 rotor. Following removal of the liquid, the invisible pellets were re-suspended with 700 μl of Qiazol reagent, and RNA was extracted as directed by the miRNEasy kit (Qiagen cat. no. 217004). Samples were denatured at 70°C and then subjected to electrophoresis on a 15% polyacrylamide TBE-urea gel 65 min at 200 V. The gel was then stained using SYBR gold and imaged with a blue light transilluminator. Gel slices were taken from the regions corresponding to lengths between 20 to 45 nt, as indicated by 10 bp ladder (Invitrogen cat. no. 10821015) and RNA size markers (5′-AUGUACACGGAGUCGAGCUCAACCCGCAACGCGA-(Phos)-3′ and 5′-AUGUACACGGAGUCGACCCAACGCGA-(Phos)-3′). The samples were then prepared as directed in Ingolia et al. (Ingolia et al., 2012), with modifications: ProtoScript II reverse transcriptase (New England Biolabs cat no. M0368) was used for reverse transcription, and different biotinylated oligonucleotides were used for the removal of E. coli rRNA sequences (Li et al., 2014). The library was sequenced on an Illumina HiSeq 2500 by the Johns Hopkins University Genetic Resources Core Facility. Each bacterial strain was processed in duplicate or triplicate.