title: Car ChIP-seq2
design: : The chromatin immunoprecipitation assay was done as in prior work using strain carR, carrying plasmid pCWU6-pCarR-3xFLAG (pcarR-3F), or empty pCWU6-p-3xFLAG (p3F) as a control. Note that pCWU6-pCarR-3xFLAG encodes CarR-3xFLAG, which is expressed from the original carR promoter. Lysates were prepared from late-log phase cultures, and incubated at 37C in an anaerobic chamber. Next, the bacterial culture was incubated with 1% formaldehyde for 20 min at 25C, and cross-linking was ceased using 0.5 M glycine. Bacterial cells were then harvested by centrifugation, washed three times with Tris-buffered saline buffer, and resuspended in a lysis buffer [10 mM Tris, 100 mM NaCl, 1 mM EDTA, 0.5 mM EDTA, 0.1% deoxycholate (pH 8.0)] containing a protease inhibitor cocktail (Sigma). The lysed cells were sonicated using Bioruptor Pico (Diagenode) for 10 cycles. Bacterial debris was removed by centrifugation for 10 min at 14 000 rpm, and a fraction of the supernatant was stored as the input sample for IP assays.   Supernatant DNA fragments ranged from 300 bp to 500 bp. Following sonication, the CarR-3xFLAG complexes were immunoprecipitated with a magnetic bead with anti-FLAG antibody (Sigma) overnight at 4C. After the ChIP reaction, samples were washed five times with an immunoprecipitation assay buffer [50 mM hydroxyethyl piperazineethanesulfonic acid buffer, 500 mM LiCl, 1 mM EDTA, 1% Non-idet P-40, and 0.7% deoxycholate (pH 7.5)] and twice with Tris-buffered saline buffer. ChIP DNA was eluted in an elution buffer (50 mM Tris-EDTA buffer, 1% EDTA, pH 7.5) at 65C for 30 min to de-crosslink. Samples were treated with RNase A and proteinase K, and ChIP DNA was then purified using the Agencourt AMPure XP PCR Purification Kit (Beckman Coulter). The purified and fragmented DNA samples were submitted to Cancer Genomics Center at The University of Texas Health Science Center at Houston (CPRIT RP240610). The 100bp-300bp DNA fragments were selected by Kapa Pure Beads (#7983280001, Roche Holding AG), which were used for library preparation. The libraries were prepared following the protocol of KAPA Hyper Prep Kit (KK8502, Roche Holding AG). Briefly, the fragmented DNAs were repaired at both ends, and an adenine (A) was added to the repaired ends. This end-repair and A-tailing procedure was performed at 20C for 30 minutes, followed by 65C for 30 minutes. Then, the adapters with unique indexes, which can identify each sample during sequencing, were ligated to both ends of each fragmented DNA. After ligation, the DNA fragments with adapters were cleaned up by magnet beads. The proper sizes of DNA fragments were selected. The selected DNA fragments were quantified by quantitative polymerase chain reaction (qPCR) and amplified by Bio-Rad C1000 Touch Thermal Cycler (Bio-Rad Laboratories). The final library was qualified by Agilent Bioanalyzer 2100 (Agilent Technologies) and quantified by qPCR. The libraries were pooled and subjected to a paired-end 75-cycle sequencing on an Illumina NextSeq 550 System using High Output Kit v2.5 (#20024907, Illumina, Inc.). Paired-end reads from two independent CarR ChIP-seq experiments were mapped to the reference with Burrows-Wheeler Alignment software BWA using the galaxy platform. MACS2 was used to identify ChIP-seq peaks where aligned reads were significantly enriched (using a q-value cut-off of 0.05) compared to a control (Input DNA). Finally, identified peaks were visually inspected, using the integrated genome browser, to confirm accurate assignment of peak centers. Average read counts were normalized according to the coverage per base for each experiment and visualized using IGV.
identifiers.namespace: SUB14833047
identifiers.id: CarCHip2