title: GSM9433458: Rep1_Mock; Enterobacteria phage T6; OTHER
library_name: GSM9433458
library_construction_protocol: Phage suspensions were lysed with 10% (w/v) SDS and 0.5 M EDTA (pH 8.0) at 68°C for 10 min, followed by the addition of 3 M sodium acetate (pH 6.5). The lysate was extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and centrifuged at 16,000 ×g for 3 min. The aqueous phase was re-extracted twice with chloroform:isoamyl alcohol, then precipitated with two volumes of ice-cold ethanol, 1/30 volume of 3 M sodium acetate (pH 6.5), and 1 μl GlycoBlue™ coprecipitant at −30°C for 20 min. The DNA pellet was washed with 75% ethanol, briefly air-dried, and resuspended in 50 μl of sterile water. High-molecular-weight genomic DNA was isolated from PEG-precipitated T6 phage lysates and adjusted to 1 µg per SITE-seq reaction. Purified DNA was incubated with 50 ng purified SegB or mock control in 20 µL reactions containing 1× NEBuffer 2 and 2 mM MnCl₂ at 30°C for 1 h. Reactions were terminated with EDTA and Proteinase K, followed by incubation at 55°C for 30 min. DNA was purified using SPRISelect beads and subjected to end repair and dA-tailing using the NEBNext Ultra II kit. Biotinylated adapter 1 oligos were annealed and ligated to end-repaired DNA using NEB Quick Ligase, with incubation at 20°C for 30 min followed by overnight incubation at 16°C. Ligation products were purified with SPRISelect beads and fragmented using NEBNext dsDNA Fragmentase at 37°C for 1 h. Fragmented DNA was purified, end-repaired, and dA-tailed again before ligation of adapter 2 using the NEBNext Ultra II Ligation Module. Adapter-ligated DNA was captured with Dynabeads in B&W buffer, washed, and amplified by PCR using Q5 Hot-Start Master Mix and recovery primers. Following magnetic separation, recovered DNA was further amplified by indexing PCR using NEBNext Multiplex Oligos for Illumina.