GSM8582203: WT RDM logarithmic rep 2 - RNA-seq; Escherichia coli; RNA-Seq
DNA samples: When resuspending frozen cell pellets, two pellets (taken from a single biological replicate) of each sample were separately resuspended with spike-in, sonicated, and then combined into one tube immediately after sonication. Individual frozen C. crescentus spike-in cell pellets were resuspended in 600 µL of 1x IPOD lysis buffer (10 mM Tris HCl, pH 8.0; 50 mM NaCl) containing 1x protease inhibitors (Roche Complete Mini, EDTA free, Roche Diagnostics GmbH, Mannheim, Germany) and 1.5 µL of ready-lyse (Epicentre, Madison, WI). The spike-in resuspension was used to resuspend one of the sample cell pellets, and then the resuspended cells were incubated for 15 minutes in a 30°C water bath. Sonication was then performed on all samples using a Branson digital sonifier with a microtip at 25% amplitude for four pulses of 5 seconds with a 5 second rest between each pulse; samples were kept in an ice/water bath during sonication. The two separate tubes for each biological sample were then combined. DNA digestion was performed by adding to the sonicated lysates 120 μg RNase A (Thermo Fisher Scientific, Waltham, MA), 12 μL DNase I (Fisher product #89835), 10.8 μL 100 mM MnCl2, and 9 μL 100 mM CaCl2, and then incubating on ice for 30 minutes. The digestion was quenched with 100 μL of 500 mM EDTA (pH 8.0), followed by clarification by centrifuge for 10 minutes at 13,000 rpm at 4°C. Aliquots were taken from the clarified lysate for IPOD-HR interface extraction, RNA polymerase chromatin immunoprecipitation, and cross-linking reversal and recovery of DNA as previously described in (Freddolino et al., 2021). The procedures described in that reference were replicated here, apart from all the 2-minute centrifuging steps instead being done in 4 minutes. For DNA recovery, standard phenol-chloroform extraction as ethanol precipitation as described in (Ausubel F, 1998), where we used Glycoblue (Ambion) as a co-precipitant, NaCl as a precipitating salt (due to the presence of SDS in our solution), and washed with ice-cold 95% ethanol to avoid loss of low molecular weight DNA and the dried DNA was resuspended in 100 μL of TEe (10 mM Tris pH 8.0; 0.1 mM EDTA pH 8.0) for input samples, 20 μL of TEe for RNAP-ChIP samples, and 50 μL of TEe for IPOD samples. RNA samples: RNA pellets were removed from -80°C and then immediately resuspended in 100 μL TE buffer (10 mM Tris pH 8.0; 1 mM EDTA pH 7.0). The resuspended pellet was treated with 1 μL lysozyme (Ready-Lyse; Lucigen, Ltd.) and incubated for 10 minutes at 4°C, followed by treatment with 10 μL proteinase K and incubation for 10 minutes at room temperature with vortexing every 2 minutes. RNA was then isolated using a Zymo RNA Clean and Concentrate 5 Kit twice for each sample, with a DNase digestion (25 μL eluate from first Zymo clean-up, 58 μL nuclease-free water, 10 μL 10X DNase Reaction Buffer, 2 μL RNase inhibitor, 5 μL Baseline Zero DNase) at 37°C for 30 minutes in-between Zymo kit clean-ups. Samples were then ribo-depleted using a NEBNext rRNA Depletion (Bacteria) Kit according to manufacturer's instructions – with the exception that 10 μL of RNA sample, containing 1 μg of RNA, was used for probe hybridization. Following ribo-depletion, samples were again cleaned-up with the Zymo Clean and Concentrate 5 Kit and then prepared for sequencing using the NEBNext Ultra II Directional RNA Sequencing Kit DNA samples: NEB Nebnext Ultra 2 DNA kit RNA samples: NEBNext Ultra 2 Directional RNA kit
GEO
GSM8582203_r1