Instrument: Illumina MiSeq
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: For RIP-seq: When the culture reached an OD600 of 2, bacteria were re-suspended in 800 µl of ice-cold lysis buffer (Tris, pH 8.0, 20 mM, KCl 150 mM, MgCl2 1 mM, DTT 1 mM), and disrupted with 1 ml glass beads (BioSpec Products, 0.1 mm diameter) by shaking at 30 Hz for 10 min. The cleared lysate obtained after centrifugation (16000 rcf, 15 min, 4°C) was incubated with 0.5 µl/OD anti-Flag antibody (Sigma, F1804) at 4°C for 30 min followed by incubation with 64 µl pre-washed Protein-A sepharose (Sigma, P6649) for another 30 min. After 5 washes with ice-cold lysis buffer, the sepharose was subjected to Phenol:Chloroform:Isopropanol extraction to isolate RNA (PCI 25:24:1, pH 4.5; Roth, X985.3). For RNA-seq: Total RNA was extracted with hot phenol and chloroform followed by DNase I treatment. For dual RNA-seq: Total RNA from the infected and sorted cells was isolated using the mirVana kit (Ambion). The samples were rRNA depleted (RNA-seq and dual RNA-seq), then poly(A)-tailed using poly(A) polymerase and the 5’triphosphate (and eukaryotic cap) structures were removed using tobacco acid pyrophosphatase (TAP). Afterwards, an RNA adapter was ligated to the 5’monophosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adapter primer and the M-MLV reverse transcriptase (NEB). The resulting cDNA was PCR-amplified to about 20-30 ng/µL using a high fidelity DNA polymerase (barcode sequences for multiplexing were part of the 3’primers). The cDNA library was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and analyzed by capillary electrophoresis (Agilent Technologies).