GSM7781803
Bacterial samples (about 100 OD) were collected by centrifugation at 6,000 rpm for 20 min at 4 °C. The bacterial pellets were resuspended in 500 µl ice-cold lysis buffer (20 mM Tris pH 8.0, 150 mM KCl, 1 mM of MgCl2, 1 mM DTT, 0.05% Tween-20), and broke with 500 µl glass beads using the Cryolys Evolution instrument, at 7,200 rpm for 15 min at 4 °C. Lysates were collected by centrifugation at 13,000 rpm for 30 min at 4 °C, and divided into new tubes equally. Cleared lysates were respectively incubated with anti-FLAG antibody (Sigma) or IgG isolate control (Invitrogen) conjugated on the protein-G magnetic beads (Thermo-Fisher) for 1 hour at 4 °C with rotation. The beads were collected by the magnet, and washed five times with 500 µl lysis buffer and resuspended in 50 µl lysis buffer. The Hfq-bound RNA was extracted by RNA clean & concentrator columns (Sangon, Shanghai). The purified RNA was resuspended in nuclease-free water. LiRIP-seq libraries were constructed using the RNAtag-seq method as described previously (Liu et al., 2023; Melamed et al., 2018), with a few modifications. Briefly, fragmented RNA was treated with DNase I and FastAP. RNA then was ligated to the 3' barcoded adaptor, and depleting Ribosomal RNA by Ribo-off rRNA depletion Kit (#N407-01, Vazyme). First strand cDNA was synthesized by HiScript II 1st Strand cDNA Synthesis Kit (#R211-01, Vazyme), and RNA was degraded after reverse transcription by 1 M NaOH and 0.5M acetic acid. The cDNA was ligated to a second adaptor. Every step needed to purify the sample with 2.5x Agencourt AMPure XP beads and 1.5x isopropanol. Libraries were constructed by PCR with Illumina P5 and P7 primers using Q5 High-Fidelity DNA Polymerase (#M0491L, NEB), and purified with 1.5x AMPure XP beads. The libraries were sequenced by 150 bp paired-end sequencing with an Illumina NovaSeq instrument.