Illumina HiSeq 2000 sequencing; Genome-Scale Mapping of Escherichia coli Ď54 Reveals Widespread, Conserved Intragenic Binding
Genome-Scale Mapping of Escherichia coli Ď54 Reveals Widespread, Conserved Intragenic Binding
Overnight cultures were started from single colonies in 2 mL M9+0.4% glycerol at 30 degrees Celsius shaking at 225 rpm and grown for ~36 hours.  40 ml subcultures were grown in M9+0.4% glycerol at 30 degrees Celsius to an OD600 of approximately 0.5 Cells were crosslinked for 20 minutes with formaldehyde (1% final concentration), pelleted by centrifugation and washed once with Tris-buffered saline (TBS). Cell pellets were resuspended in 1 ml FA lysis buffer (50 mM Hepes-KOH, pH 7, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) with 4 mg/ml lysozyme and incubated at 37 degrees Celsius for 30 minutes. Samples were then chilled and sonicated for 30 minutes in a Bioruptor sonicator (Diagenode) with 30 s on/30 s off pulsing at maximum amplitude. Samples were pelleted in a microcentrifuge to remove debris and supernatants (chromatin) were saved, 2.5 ml FA lysis buffer was added, and samples were stored indefinitely at -20 degrees Celsius. For the immunoprecipitation (IP), 800 ul chromatin was incubated with 25 ul Protein A Sepharose slurry (50%) in TBS and 5 ul anti-sigma 54 antibody (BioLegend) for 90 minutes at room temperature with gentle mixing on a rotisserie rotator.  Following the 90 min incubation, beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 7.5 (please note: all wash steps involved incubation of samples with the wash solution for 3 min with gentle mixing on a rotisserie rotator before pelleting beads at 1,500 x g in a microcentrifuge for 1 min).  Beads were resuspended in 100 uL 1X Quick Blunting Buffer (NEB) containing dNTPs (concentration specified by NEB Quick Blunting kit) and 1 uL Quick Blunting enzyme mix (NEB), and incubated at room temperature for 30 min with gentle mixing. Beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 8.0. Beads were resuspended in 100 uL Buffer 2 (NEB) containing 2 mM dATP and 2 uL Klenow DNA polymerase (NEB), and incubated for 30 min at 37 degrees Celsius with gentle mixing. Beads were washed twice with FA lysis buffer and twice with 10 mM Tris-HCl, pH 7.5. Beads were resuspended in 1X DNA Quick Ligase buffer (NEB), 1 uL NEXTflex ChIP-seq Barcoded adapter (BioO) and 2 uL Quick DNA Ligase (NEB), and incubated at room temperature for 15 min with gentle mixing. Beads were washed twice with FA lysis buffer, and once each with high salt FA lysis buffer (50 mM Hepes-KOH, pH 7, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), ChIP wash buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% Nonidet-P40, 0.5% sodium deoxycholate) and TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). After the TE wash, beads were transferred to a fresh Spin-X column and eluted with 100 ul ChIP elution buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) for 10 minutes at 65 degrees Celsius with occasional agitation. Samples were decrosslinked by boiling for 10 minutes. DNA was extracted with phenol/chlorofom/isoamyl alcohol, ethanol precipitated, and resuspended in 11 uL H2O. 1 uL DNA was diluted 1:8 and 2 ul were used for real time PCR amplification (three technical replicates)with NEXTflex ChIP-seq Primer Mix (JW3764-AATGATACGGCGACCACCGAGATCTACAC and JW3765-CAAGCAGAAGACGGCATACGAGAT)  using an ABI 7500 Fast real time PCR machine. The number of cycles required to reach a Delta Rn score of 0.1 was recorded (OXO). 8 uL of the remaining DNA was amplified using conventional PCR with oligonucleotides JW3764 and JW3765 ( 100 uM) for X+3 cycles with an annealing temperature of 60 degrees Celsius. PCR products were purified using Ampure XP magnetic beads (Ampure) and resuspended in 20 uL H2O. Purified PCR products between 200 bp and 600 bp were gel purified from an 8% non-denaturing polyacrylamide gel, eluted overnight in DNA extraction buffer (0.3 M NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA) and ethanol precipitated to generate the final ChIP-seq library. The library was resuspended in 11 uL H2O and quantified using a Qubit fluorimeter (Invitrogen). The library was sequenced using a HiSeq 2000 sequencer (Illumina; University at Buffalo Next Generation Sequencing Core Facility).