GSM8977091: Hfq-R16A, ctrlplasmid, IP, 1; Escherichia coli; OTHER
The cells were lysed by vortexing with 212-300 mm glass beads (Sigma) in a total volume of 2 ml lysis buffer (20 mM Tris-HCl/pH 8.0, 150 mM KCl, 1 mM MgCl2 and 1 mM DTT). Isolation of total RNA and Hfq co-IP RNA was performed as described previously. 1.9 ml of cell lysate were mixed with 100 µl of Hfq antibody and 120 mg of protein A-Sepharose CL-4B (GE Healthcare). Hfq co-IP RNA was isolated from protein A-Sepharose beads by extraction with phenol:chloroform:isoamyl alcohol (25:24:1), followed by ethanol precipitation. Total RNA was isolated from 100 µl of cell lysate by Trizol (ThermoFisher Scientific) extraction followed by chloroform extraction and isopropanol precipitation. RNA was dissolved in 15 µl of DEPC water. cDNA libraries were constructed based on a protocol adapted to capture the bacterial sRNAs as well as mRNAs (Melamed et al., 2018, Nat. Protoc., 13, 1-33). 400 ng of total RNA or Hfq co-IP RNA was used as input for cDNA synthesis, with total RNA serving as input for Hi-GRIL-seq and Hfq co-IP RNA for iRIL-seq. Ribosomal RNA was depleted using DASH protocol. 12 cycles of PCR were performed at the last step to enrich each library.