title: GSM8335783: KpnDRT2_SL2_MUT_input; Escherichia coli str. K-12 substr. MG1655; RIP-Seq
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 RIP elution, the supernatant was removed, and beads were resuspended in 750 µL TRIzol (Thermo Fisher Scientific). After 5 min incubation at RT, the supernatant containing eluted RNA was transferred to a new tube and combined with 150 μL chloroform. Samples were mixed vigorously by inversion, incubated at RT for 3 min, and centrifuged at 12,000 x g for 15 min at 4 °C. RNA was isolated from the upper aqueous phase using the RNA Clean & Concentrator-5 kit (Zymo Research) and eluted in 15 μL RNase-free water. RNA from input samples was isolated in the same manner using TRIzol and column purification. Purified RNA was stored at -80 °C before proceeding to library preparation. For RIP-seq library preparation (input and RIP eluates), RNA was fragmented by random hydrolysis by combining 7 µL RNA, 6 µL water, and 2 µL NEBuffer 2, and heating to 92 °C for 2 min. To remove DNA and prepare RNA ends for adapter ligation, samples were treated with 2 μL TURBO DNase (Thermo Fisher Scientific) and 2 μL RppH (NEB) in the presence of 1 µL SUPERase•In RNase Inhibitor for 30 min at 37 °C. This was followed by treatment with 1 µL T4 PNK (NEB) in 1× T4 DNA ligase buffer (NEB) for 30 min at 37 °C. Reactions were column-purified using the Zymo RNA Clean & Concentrator-5 kit and eluted in 10.5 μL RNase-free water. RNA concentrations were quantified using the DeNovix RNA Assay. Illumina sequencing libraries were prepared using the NEBNext Small RNA Library Prep kit, and libraries were sequenced on an Illumina NextSeq 500 in paired-end mode with 150 cycles per end.