GSM7957473: MaAgo-associated siDNA, with MaACE1; Escherichia coli; OTHER
To extract nucleic acids co-puriﬁed with MaAgo, 40 µl of purified protein was mixed with 310 µl of amylose buffer supplemented with EDTA (10 mM ﬁnal concentration) and 40 µl proteinase K (20 mg/ml, New England Biolabs). The sample mix was incubated for two hours at room temperature. After incubation, 400 µl Roti phenol/chloroform/isoamyl alcohol was added, the sample was vortexed, and centrifuged for 15 min. The upper aqueous phase was isolated and 0.1 volumes of 1M sodium acetate, 3 volumes 100% ethanol, and 10 µl of linear polyacrylamide (Alfa Aesar) were added. This mix was briefly vortexed and incubated at -20 °C for 16 h. The samples were centrifuged for 30 min, after which the supernatant was removed without disturbing the pellet. The pellet was washed with 500 µl 70% ethanol (pre-cooled to -20 °C) and centrifuged for 15 min. Without disturbing the pellet, the ethanol was removed. The pellets containing the copuriﬁed nucleic acids were further dried for 10 min at 50 °C, and were resuspended in 50 µl of RNase free water. The DNA extracted from purified MaAgo was treated with RNase A (Thermo Scientific) according to the protocol of the manufacturer. The remaining DNA was puriﬁed using PCI extraction, and treated with T4 PNK (Thermo Fisher, EK0031) according to the protocol of the manufacturer. Subsequently, the DNA was again purified using PCI extraction and used as input for library preparation using the protocol described in Olovnikov, et al. (https://pubmed.ncbi.nlm.nih.gov/24034694/)