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. Author manuscript; available in PMC: 2014 Sep 15.
Published in final edited form as: Methods. 2013 May 23;63(2):144–159. doi: 10.1016/j.ymeth.2013.04.023

Figure 2. Discovery of Hfq Binding RNAs.

Figure 2

A. Co-IP of Hfq bound RNAs using chromosomally FLAG tagged Hfq (adapted from Sittka et al.) [36]. Cellular extracts from hfqFLAG cells are prepared and co-IP with an α-FLAG antibody is performed. RNAs are extracted and modified to incorporate a polyA tail and 5'phosphate. A 5' adapter is ligated followed by cDNA synthesis and high-throughput sequencing to identify the bound RNAs. B. Genomic SELEX (adapted from Lorenz et al. and Zimmermann et al.) [68, 130]. A genomic library is created by random priming using primers that incorporate a T7 promoter and primer binding sites for reverse transcription and PCR. The library is transcribed to RNA and a binding reaction with Hfq is performed. Bound complexes are selected using filter binding. Bound RNAs are recovered from the filter followed by RT-PCR. The cycle is repeated multiple times followed by sequencing to identify the aptamers.