title: GSM3983606: InpWT; Vibrio cholerae O1 biovar El Tor str. N16961; ChIP-Seq
library_construction_protocol: The cell pellets were resuspended in 1 ml buffer CJ2 [100 mM Na2HPO4, 600 mM NaCl, 0.05% tweenTM, and 30 mM imidazole;pH 8.0] after washing twice with same CJ1 buffer. Cells were then lysed by sonication, followed by addition of Micrococcal nuclease (100 U ml−1, NEB cat# M0247S) and RNase A (1 mg ml−1) for fragmentation of genomic DNA and removal of RNA. 950 μl of supernatant was collected after centrifugation. For DNA-VxrB complex purification, HisPurTM Ni-NTA magnetic beads (cat#88831-Thermo Scientific) were equilibrated with six bed volumes of CJ2 buffer [100 mM Na2HPO4, 600 mM NaCl, 0.05% tweenTM, and 30 mM imidazole;pH 8.0]. 500 μg of crude lysate (protein concentration determined by Bradford assay) was mixed with the Ni-NTA resin and incubated at 4°C for 4 hours on rotation mixer. The bead slurry was recovered via magnetic stand for 5 min standing and washed twice with 400 μl buffer CJ3 [100 mM Na2HPO4, 600 mM NaCl, 0.05% tweenTM, and 50 mM imidazole; pH 8.0]. The VxrB-DNA complexes were then eluted from the beads by addition of CJ4 elution buffer [100 mM Na2HPO4, 600 mM NaCl, 0.05% tweenTM, and 500 mM imidazole;pH 8.0]. Enriched target DNAs were purified by using PCR Clean-Up Kits (Thermo Fisher Scientific) after reversal of crosslinking at 65°C overnight. The desired DNA fragment size (200~300 bps) was then purified by gel extraction. For illumina HiSeq, each DNA library was constructed with specific barcode sequences, according to the manufacturer's instruction of TruSeq ChIP Library Preparation Kit (IP-202-1012, Illumina). The constructed libraries were sequenced using 100-bp paired-end read protocol on an Illumina HiSeq 2500 platform (Illumina) at the Biotechnology Resource Center (BRC) at Cornell University.
attributes.tag: GEO Accession
attributes.value: GSM3983606