GSM8140823: C. botulinum dTnpB Input; Escherichia coli; RIP-Seq
10 mL of culture was centrifuged at 4,000 g for 10 min at 4 °C, and the supernatant was removed. The pellet was washed once with 1 mL of cold TBS (20 mM Tris-HCl (pH 7.5 at 25 °C), 150 mM NaCl), centrifuged at 10,000 g for 5 min at 4 °C, the supernatant was removed, and the resulting pellet was flash-frozen in liquid nitrogen. Pellets were stored at -80 °C. Antibodies for immunoprecipitation were conjugated to magnetic beads as follows: for each sample, 30 μL Dynabeads Protein G (Thermo Fisher Scientific) were washed 3× in 1 mL RIP 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 RIP lysis buffer, combined with 10 μL anti-FLAG M2 antibody (Sigma-Aldrich, F3165), and rotated for >3 hours at 4 °C. Antibody-bead complexes were washed 3× to remove unconjugated antibodies, and resuspended in 30 μL RIP lysis buffer per sample. To generate cell lysates, flash-frozen pellets were first resuspended in 1.2 mL RIP lysis buffer supplemented with cOmplete Protease Inhibitor Cocktail (Roche) and SUPERase•In RNase Inhibitor (Thermo Fisher Scientific). Cells were then sonicated for 1.5 min total (2 s ON, 5 s OFF) at 20% amplitude. To clear cell debris and insoluble material, lysates were centrifuged for 15 min at 4 °C at 21,000 g, and the supernatant was transferred to a new tube. At this point, a small volume of each sample (24 μL, or 2%) was set aside as the “input” starting material and stored at -80 °C.For immunoprecipitation, each sample was combined with 30 μL antibody-bead complex and rotated overnight at 4 °C. The next day, each sample was washed 3× with ice-cold RIP wash buffer (20 mM Tris-HCl (pH 7.5 at 25 °C), 150 mM KCl, 1 mM MgCl2). After the last wash, beads were resuspended in 1 mL TRIzol (Thermo Fisher Scientific) and incubated at RT for 5 min to allow separation of RNA from the beads. A magnetic rack was used to isolate the supernatant, which was transferred to a new tube and combined with 200 μL chloroform. Each sample was mixed vigorously by inversion, incubated at RT for 3 min, and centrifuged for 15 min at 4 °C at 12,000 g. 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. For RIP-seq library preparation (input and RIP eluates), 6 μL of RNA was diluted in FastAP Buffer (Thermo Fisher Scientific) supplemented with SUPERase•In RNase Inhibitor (Thermo Fisher Scientific) to a total volume of 18 μL, and fragmented by heating to 92 °C for 1.5 min. Each sample was treated with 2 μL TURBO DNase (Thermo Fisher Scientific) for 30 min at 37 °C and column-purified using the RNA Clean & Concentrator-5 kit (Zymo Research), eluting in 12.5 μL RNase-free water. RNA concentration was 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 75 cycles per end.