title: GSM9564323: CasPRIB1-his6 (input) biol rep2; Listeria seeligeri; ChIP-Seq
library_construction_protocol: 10 min 1% formaldehyde, glycine quench. Cultures were pelleted via centrifugation at 4C and washed 3 times by resuspension in 10 mL ice cold 1X PBS. After washes, pellets were resuspended in 1.5 mL  lysis buffer (50 mM HEPES pH 7.0, 150 mM NaCl, 5 mM MgCl2, 10 mM imidazole, 1 mg/mL lysozyme, 1 mM PMSF, 5% glycerol), and incubated at 37C for 15 min. Samples were sonicated (Qsonica Q500) on ice using 15% amplitude in 10 s intervals with a 50% duty cycle for 10 minutes of processing time, which resulted in DNA fragments averaging 500 bp.  Processed lysates were centrifuged for 10 minutes at 4C. Here, 50 μL of clarified lysates were reserved as “total DNA” matched pairs for each sample. Remaining supernatants were applied to 100 μL of equilibrated Ni-NTA agarose resin (ThermoFisher) and incubated at 4C with rotation for two hours. Then, mixtures were centrifuged at 4C and 2000 rpm for 1 min to pellet beads. Beads were washed with 1 mL of cold wash buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 5 mM MgCl2, 10 mM imidazole, 5% glycerol) and further incubated at 4C for 5 min. This was repeated twice for three total washes. After the final wash, 50μL elution buffer (20 mM HEPES pH 7.0, 150 mM NaCl, 5 mM MgCl2, 250 mM imidazole, 5% glycerol) was added to each bead pellet. Samples were incubated at RT for 5 minutes and centrifuged at 4C and 2000 rpm for 1 min before carefully harvesting the IP supernatant. Total DNA and IP samples were incubated at 60C overnight to reverse crosslinks. After overnight incubation, 450 μL of wash buffer was added to each sample and samples were treated with RNase A (0.2 mg/mL) at 37C for 1 h and subsequently with Proteinase K (0.2 mg/mL) at 37C for 30 min. Next, an equal volume of phenol/chloroform (1:1) was added to each sample and vortexed before centrifuging samples at RT for 10min at 13,000 rpm. The aqueous phase of each sample was then harvested and combined with 50 μL sodium acetate. DNA was precipitated with two volumes of cold ethanol and pelleted by centrifugation (15,000 rpm) at 4C for 15min. Pellets were washed two times with 70% ethanol before resuspending IP samples in 10 μL water and total DNA samples in 50 μL water. DNA fragments for each sample were barcoded and prepared for sequencing using the Illumina TruSeq DNA Library Preparation kit according to the manufacturer's instructions. Purification steps were performed using Ampure beads and samples were pooled equally by weight to form final libraries. CasPR ChIP-seq libraries were sequenced by Azenta on the Illumina NextSeq platform (2x150 bp reads). RpoC-his10 libraries were sequenced on the Illumina MiSeq i100 platform (2x50 bp reads).