Criterion 10 polyacrylamide tbe urea gel

Linear DNA fragments containing the T7 promoter binding site followed by the 20-bp sgRNA target sequence were transcribed in vitro using the T7 High Yield RNA Synthesis Kit (NEB) according to the manufacturer’s instructions. In vitro transcribed RNA was precipitated with ethanol and purified by gel electrophoresis on a Criterion 10% polyacrylamide TBE-Urea gel (Bio-Rad). Excised gel fragments were extracted in 420 µL of 300 mM NaCl overnight on a rocking surface at 4 °C. Gel-purified sgRNA was precipitated with ethanol and redissolved in water and sgRNA concentration was finally quantified by UV absorbance and snap-frozen at −80 °C.

Full-length λ dsDNA (NEB; 48 502 bp) was prepared as previously described (14 (link)). Briefly, λ dsDNA was hybridized and ligated to an oligonucleotide, complementary to the ssDNA tail at one of its ends, and containing a Digoxigenin-labeled oligonucleotide and to the ssDNA tail with a biotinylated oligonucleotide at the other end.
Total RNA was extracted from exponentially growing culture of Saccharomyces cerevisiae strain YS1267 using TRIzol (Invitrogen) and following the manufacturer's instructions. Other RNAs (Figure 1) were transcribed in vitro using T7 Quick High Yield RNA Synthesis Kit (NEB) and DNA templates prepared by amplification with Crimson LongAmp^® Taq DNA polymerase (NEB). For short transcripts (<0.3 kb), incubation of amplification reactions was extended to 24 h. Long RNAs were purified by phenol-chloroform extraction and ethanol precipitation, and dissolved in DEPC-treated water. Short RNAs (130 and 416 nt) were purified by gel electrophoresis on a Criterion 10% polyacrylamide TBE–urea gel (Bio-Rad), precipitated with ethanol and redissolved in DEPC-treated water. The shortest RNA (40 nt) and other oligonucleotides used in this study were purchased from Integrated DNA Technologies. Oligonucleotide sequences are listed in Supplementary Table S1.