GSM3701634: PP7_invitro.PP7_14_AGTTCC_L001; Escherichia coli; Pseudomonas phage PP7; OTHER
RNA isolation of 5S rRNA was performed using TRIzol-based standard protocols. Briefly, cells were lysed using Max Bacterial Enhancement Reagent followed by TRIzol treatment (both from Life Technologies). Phase separation was performed using chloroform. RNA was precipitated from the aqueous phase using isopropanol and ethanol washes, and then resuspended in RNase-free water. For the strains harboring PP7-wt δ=-29 and PP7-USs δ=-29, column-based RNA isolation (RNeasy mini kit, QIAGEN) was performed. To produce the dsDNA for sequencing 10ul of purified cDNA from the SHAPE procedure (see above) were PCR amplified using 3 primers: 4nM mCherry selection (#4) or 5S rRNA selection primer (#5), 0.5µM TruSeq Universal Adapter (#6) and 0.5µM TrueSeq Illumina indexes (one of #7-26) (Table S3) with PCR reaction mix (1x Q5 HotStart reaction buffer, 0.1 mM dNTPs, 1 U Q5 HotStart Polymerase [NEB]). A 15-cycle PCR program was used: initial denaturation at 98°C for 30 s followed by a denaturation step at 98°C for 15 s, primer annealing at 65°C for 30 s and extension at 72°C for 30 s, followed by a final extension 72°C for 5 min. Samples were chilled at 4°C for 5 min. After cool-down, 5 U of Exonuclease I (ExoI, NEB) were added, incubated at 37°C for 30 min followed by mixing 1.8x volume of Agencourt AMPure XP beads to the PCR/ExoI mix and purified according to manufacturer's protocol. Samples were eluted in 20 µl ultra-pure water. After library preparation, samples were analyzed using the TapeStation 2200 DNA ScreenTape assay (Agilent) and the molarity of each library was determined by the average size of the peak maxima and the concentrations obtained from the Qubit fluorimeter (Thermo Fisher Scientific). Libraries were multiplexed by mixing the same molar concentration (2-5 nM) of each sample library, and library and sequenced using the Illumina HiSeq 2500 sequencing system using either 2X51 paired end reads for the 5S-rRNA control and in vitro experiments or 2x101 bp paired-end reads for all other samples. SHAPE-seq
GEO Accession
GSM3701634