title: ssDNA sequencing of DRT2-expressing E. coli: T5-infected cells, YCAA mutant
design: DNA (500 ng) was treated with and without 100 U S1 Nuclease (Thermo Scientific) in 1 reaction buffer at 25C for 2h before dilution with 50 mM Tris-HCl pH 7.5, extraction with phenol-chloroform-isoamylalcohol (25:24:1), and ethanol precipitation. Half of the precipitated DNA was used as input for strand-specific DNA libraries. Input DNA was supplemented with 1 fmol of 200 nt control ssDNA (ssd_spike) and incubated with 20 U terminal transferase (NEB) and 4 M dATP in 1 reaction buffer (without CoCl2) at 37C for 30 min then 70C for 5 min. 2.5 pmol of primer ssExt_pT9_anchor was then annealed to the DNA, followed by addition of 1 mM dNTPs and 5 U Klenow Fragment (exo-, NEB). The reaction was incubated at 37C for 30 min then 65C for 5 min, and products were purified using the QIAquick PCR purification kit (Qiagen). Adapters ssd_adapt_top and ssd_adapt_bottom were annealed, and 100 fmol was ligated to 13% of the recovered DNA using Blunt/TA Ligase Master Mix (NEB). Ligation reactions were amplified with PhusionFlash PCR master mix and barcoded primers for 20 cycles. Amplicons larger than the 147 bp adapter dimer were purified by gel extraction and sequenced on a MiSeq in single-end mode (290 bp read 2).