GSM5123242: sm_BL_kM_pET_rep2; Escherichia coli; OTHER
All purification steps were carried out at 4˚C. The cell pellet was resuspended in lysis buffer (50 mM Tris-HCl, 250 mM NaCl, 5% glycerol, 0.5 mM β-mercaptoethanol, pH 7.4 at 23˚C) supplemented with EDTA-free protease inhibitor cocktail (Roche) at approximately 7 ml per gram of wet weight and subjected to 3 rounds of disruption on a high-pressure homogenizer (Avestin) at 12000 psi. The cell lysate was clarified for 30 min at 35,000 g. KmAgo was pulled down on Talon metal affinity resin (Takara) charged with Co 2+ for 2 hours at 4˚C (300 ul of bead suspension per sample) with constant rocking. The beads were collected by centrifugation (300 g, 3 min) and washed one time with lysis buffer and three times with lysis buffer supplemented with 10 mM imidazole (140 ml in total). KmAgo-smDNA complexes were eluted in 500 ul of lysis buffer supplemented with 300 mM imidazole. Nucleic acids associated with KmAgo were extracted with phenol:chloroform:isoamyl alcohol (PCI) mixture (25:24:1, pH 8.0) according to a standard procedure. Briefly, the protein sample was mixed with an equal volume of PCI, vortexed intensively for 30 seconds and centrifuged at 13000 g for 3 min. The upper aqueous phase was carefully collected and treated twice with an equal volume of chloroform to eliminate any remaining traces of phenol. Nucleic acids were precipitated with three volumes of ethanol in the presence of a co-precipitating agent (PINK, Bioline) and dissolved in 20 ul of milliQ-grade water. Libraries for high throughput sequencing of smDNAs were prepared according to the previously described bridged (splinted) ligation protocol. For the purpose of visualization, a 5 ul aliquot of the smDNA sample (1/4 of total volume) was dephosphorylated and radiolabeled as described above. Unlabeled DNA was mixed with the radiolabeled aliquot and 2x urea sample buffer (8 M urea, 20 mM EDTA, 0.005% Bromophenol Blue, 0.005% Xylene Cyanol and resolved under denaturing conditions in a 15% polyacrylamide-urea gel. Gel bands corresponding to 14-25 nt smDNAs were crashed and DNA was eluted in 0.4 M NaCl solution overnight at 21˚C with constant agitation. After ethanol precipitation, DNA was resuspended in 20 ul of milliQ-grade water. Illumina-compatible adaptors were ligated to both ends of smDNA. For this purpose, 8 ul of 5 x Rapid Ligation buffer (ThermoFisher), 2 ul of 100 uM 5'-adaptor, 2 ul of 100 uM of 3'-linker, 2 ul of 100 uM oligonucleotide bridge 1, 2 ul of 100 uM oligonucleotide bridge 2 and 2 units of T4 PNK (ThermoFisher) were added to 20 ul of the purified smDNA solution. The reaction mixture was incubated overnight at room temperature. Ligated DNA fragments were recovered by 10% denaturing PAGE. The libraries were amplified and indexed according to the standard protocol (small RNA sequencing kit, NEB), except that PCR conditions were adjusted in order to prevent library over amplification. A series of test PCR were performed to optimize the amount of adaptor-ligated smDNA to achieve the desired amplification level in 3-4 cycles. smDNA libraries were sequenced using the HiSeq2500 platform (Illumina) in the rapid run mode (50 nt single-end reads).