GSM4151182: Hfq65_Total_Rep1; Escherichia coli K-12; RNA-Seq
Polyclonal antibodies to Hfq were generated previously by immunizing rabbits with purified Hfq protein (Covance). RNAs that co-IP with Hfq were isolated as described previously(Zhang, A., et al., Mol Cell, 9, 11-22) with the following modifications. Overnight cultures of strains KK2440 (WT) and KK2448 (Hfq65) cells were grown to OD600 ~ 1.0 in LB medium. Cells corresponding to the  equivalent of 40 OD600 were collected, and 2 ml cell lysates were prepared by vortexing with 212-300 μm glass beads (Sigma) in a final volume of 2 ml lysis buffer (20 mM Tris-HCl/pH 8.0, 150 mM KCl, 1 mM MgCl2, 1 mM DTT). Co-IPs were carried out either using 200 μl of α-Hfq, 240 mg of protein A-Sepharose CL-4B (GE Healthcare), and 1.9 ml of cell lysate. 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 (Thermo Fisher Scientific) extraction followed by chloroform extraction and isopropanol precipitation. Total and co-IP RNA samples were resuspended in 30 μl and 50 μl of DEPC H2O.  The integrity of RNA was checked on 1% agarose gel. The RNA were treated with DNAase I(100 μl) followed by phenol: Chloroform: IAA based purification and ethanol precipitation. The gDNA contamination was checked by PCR amplification.  The DNA free RNA samples were then subjected to rRNA depletion using Ribo-zero kit and purified by using (2.5X)RNAClean XP Beads and isopropanol (1.5X). isopropanol ratio(1.5X) is critical to keeping short RNAs (>70 nt) om the RMA [pp;. The isopropanol and RNAClean XP bead ratios were both optimized in orger to remove nonligated adaptors while saving short RNA fragments. The RNAs (400 ng) were used for cDNA library preparation.  Library construction was carried out based on the RNAtag-Seq methodology (Shishkin, et al. 2015, Nat Methods, 12, 323-325), which was adapted to capture (optimized amount of isopropanol and RNAClean XP for short sRNA) bacterial sRNA (Melamed, S et al.,  2018, Nat Protoc, 13, 1-33).   To enrich the libraries, 12 cycles of PCR were carried out at the end step of enrichment. Bead purification was performed twice at the last step to ensure high purity of the library from the adaptor dimer.