GSM8268680: HrAgo1-associated small RNAs; Escherichia coli; ncRNA-Seq
To extract nucleic acids co-puriﬁed with HrAgo1, 2 nmoles of purified protein was incubated with 250 µg/ml proteinaseK (Thermo Scientific) for 4h at 65C. Next, phenol:chloroform:IAA 25:24:1 pH 7.9 (Invitrogen) was added in a 1:1 ratio.  The sample was vortexed and centrifuged at 16000 x g in a table top centrifuge for 10 min. The  upper layer containing the nucleic acids was transferred to a clean tube and the nucleic acids were  precipitated through ethanol precipitation. To this end, 99% cold ethanol and 3 M sodium acetate  pH 5.2 were added to the sample in a 2:1 and 1:9 ratio, respectively. The sample was incubated  overnight at -80°C, after which it was centrifuged at 16000 x g in a table top centrifuge for 1 h. The  pellet was washed with 70% ethanol and subsequently dissolved in nuclease-free water. The small RNAseq library was constructed by GenomeScan (Leiden, NL) using standard small RNA library preparation protocols