smDNA from pBAD LrAgo E. coli BL21
Overnight bacterial cultures were obtained from frozen aliquoted cultures and inoculated into 500 ml of fresh LB medium supplemented with 0.01% L-arabinose and ampicillin (100 g/ml) in 2l flasks. The flasks were incubated at 190 rpm at 30C in an orbital shaker and cell density was monitored by measuring OD600 every 30 min. Cells were harvested by centrifugation at 7,000g, 4C for 15 min after 5.5 and 12.5 hours of growth for protein pull-down.  The cells were disrupted with a high-pressure homogenizer (EmulsiFlex-C5, Avestin) at 18000 psi. pAgos were pulled down using Co2+-Talon Metal Affinity Resin (Takara) as described previously (Olovnikov, 2013). Eluted proteins were treated with Proteinase K for 30 minutes at 37C, small nucleic acids were extracted with phenol-chloroform, ethanol-precipitated, dissolved in water, treated with DNase I or RNase A and analyzed by PAGE as described (Olovnikov, 2013).  Libraries for high-throughput sequencing of small DNAs were prepared according to the previously published splinted ligation protocol (Olovnikov, 2013). Briefly, nucleic acids extracted from pAgos were treated with RNase A (Thermo Fisher), purified by PAGE, small DNAs (14-20 nt) were eluted from the gel in 0.4 M NaCl overnight at 21 C, ethanol precipitated, dissolved in water, phosphorylated with polynucleotide kinase (New England Biolabs), and ligated with adaptor oligonucleotides using bridge oligonucleotides as described in (Olovnikov, 2013). The ligated DNA fragments were purified by denaturing PAGE, amplified, and indexed by the standard protocol for small RNA sequencing (New England Biolabs). Small DNA libraries were sequenced using the HiSeq2500 platform (Illumina) in the rapid run mode (50-nucleotide single-end reads).