title: GSM8335738: KpnDRT2_WT_rep1_infected_input; Escherichia coli str. K-12 substr. MG1655; OTHER
library_construction_protocol: Cells were harvested by centrifugation at 4,000 x g for 10 min at 4 °C, and the supernatant was removed. The pellet was washed with 5 mL of cold TBS (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM NaCl) and spun down again as before. The supernatant was removed, and the pellet was washed with 1 mL of cold TBS before centrifugation at 10,000 x g for 5 min at 4 °C. The supernatant was removed, and the pellet was flash-frozen in liquid nitrogen and stored at -80 °C. Antibodies for immunoprecipitation were conjugated to magnetic beads as follows: for each sample, 60 μL Dynabeads Protein G (Thermo Fisher Scientific) were washed 3× in 1 mL IP lysis buffer (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM KCl, 1 mM MgCl2, 0.2% Triton X-100), resuspended in 1 mL IP lysis buffer, combined with 20 μL anti-FLAG M2 antibody (Sigma-Aldrich, F3165), and rotated for > 3 hours at 4 °C. Antibody-bead complexes were washed 2× to remove unconjugated antibodies and resuspended in 60 μL of IP lysis buffer per sample. Flash-frozen pellets were thawed on ice and resuspended in 1.2 mL IP lysis buffer supplemented with 1× cOmplete Protease Inhibitor Cocktail (Roche) and 0.1 U µL−1 SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). To lyse cells, samples were sonicated using a 1/8″ sonicator probe for 1.5 min total (2 s ON, 5 s OFF) at 20% amplitude. To clear cell debris and insoluble material, lysates were centrifuged at 21,000 x g for 15 min at 4 °C, and 1 mL supernatant was transferred to a new tube. At this point, 10 µL of each sample were set aside as “input” starting material and stored at -80 °C. For immunoprecipitation, each sample was combined with 60 μL antibody-bead complex and rotated overnight at 4 °C. The next day, each sample was washed 3× with 1 mL ice-cold IP wash buffer (20 mM Tris-HCl, pH 7.5 at 25 °C, 150 mM KCl, 1 mM MgCl2), using a magnetic rack to immobilize the beads in between each wash. For cDIP elution, the supernatant was removed, and beads were resuspended in 90 µL IP wash buffer and treated with 5 µg RNase A (Thermo Fisher Scientific) for 30 min at 37 °C. Input samples were adjusted to 90 µL with IP wash buffer and treated with RNase A in parallel. SDS was added to IP and input samples to a final concentration of 1%, and samples were treated with 25 µg Proteinase K (Thermo Fisher Scientific) for 30 min at 55 °C. Beads were immobilized using a magnetic rack, and the supernatant containing eluted DNA was transferred to a new tube. DNA was isolated using the Monarch PCR and DNA Cleanup kit (NEB), following the Oligonucleotide Cleanup protocol and eluting in 15 µL DNase-free water. For Retron-Eco1 samples, DNA was treated with DBR1 (Origene) in reactions containing 2 µL DNA, 0.5 µL DBR1, 1× rCutSmart in 10 µL total volume, in order to cleave the 2′-5′ phosphodiester linkage between msRNA and msDNA. Reactions were cleaned up using the Monarch PCR and DNA Cleanup kit (NEB), with elution in 15 µL DNase-free water. Purified DNA was stored at -80 °C before proceeding to library preparation. For cDIP-seq library preparation, 2 µL of each input sample and 10 µL of each IP eluate were diluted to 15 µL with DNase-free water. Samples were denatured by heating at 95 °C for 2 min, and then immediately placed on ice. Ligation of Illumina adapters and conversion of ssDNA to dsDNA were performed using the xGen ssDNA & Low-Input DNA Library Prep Kit (IDT), and libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 150 cycles per end.