Computational protocol: Regulation of the Na+/K+-ATPase Ena1 Expression by Calcineurin/Crz1 under High pH Stress: A Quantitative Study

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Protocol publication

[…] ChIP experiments were performed as described previously []. The biological material for ChIP-Seq was prepared essentially as for ChIP with the following exceptions: i) chromatin was fragmented using a Bioruptor Plus UCD-300 equipment (Diagenode) provided with a cooling system (4°C) for 30 cycles (high intensity; 30 s of sonication followed by 60 s pause) to generate fragments of ≤ 500 bp length; ii) the supernatant was pre-cleared with protein G sepharoseTM fast flow (GE Healthcare, #17-0618-01) beads for 1 h at 4°C and then, the pre-cleared cell lysate was incubated with 1 μg of polyclonal anti-HA ChIP-grade (Abcam, #ab9110) antibody overnight at 4°C; iii) anti-HA-Crz1-DNA complexes were collected with protein-G-sepharose beads incubating for 1 hour at 4°C and iv) after reverse crosslinking, DNA was extracted using a NucleoSpin® Gel and PCR Clean-up kit (Macherey Nagel, #740609) and NTB buffer (Macherey-Nagel, #740595); v) recovered DNA (40 ng/reaction) was subjected to PCR using oligonucleotides that recognize various regions of ENA1 promoter sequences. Oligonucleotide primer sequences are given in . ChIP libraries were prepared using the TruSeq ChIP Sample Preparation Kit (Illumina) and then subjected to paired-end deep sequencing with the MiSeq Reagent Kit v2 (300 cycle) to provide reads of around 150 nt. FASTQ files were mapped using Bowtie2 (version 2.1.0) to generate the corresponding SAM files. Mapped reads (1.9 to 3.7 million/time point) were sorted and indexed using IGV tools []. Subsequent analysis was performed using the SeqMonk software (Babraham Institute, http://www.bioinformatics.babraham.ac.uk/projects/seqmonk/). The S. cerevisiae EF4 genomic data was employed. The chromosomal coordinates of the intergenic regions and information on the flanking genes were obtained from the Saccharomyces Genome Database using the YeastMine tool. This information was loaded into SeqMonk as an Annotation set and was used to generate 6303 intergenic probes. Subsequently, each of these probes were tiled in contiguous sections of 50 nt. This generated a total number of 65198 probes that were quantified by the read count quantification method, with identical reads removed and correction for total read count referred to the largest Dataset. […]

Pipeline specifications

Software tools Bowtie2, IGV, SeqMonk
Application ChIP-seq analysis
Organisms Saccharomyces cerevisiae
Chemicals Calcium, Potassium, Sodium