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| Status |
Public on Jan 02, 2018 |
| Title |
exp2_Input_Vehicle |
| Sample type |
SRA |
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| Source name |
mpkCCD cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: cultured mouse cortical collecting duct cells (mpkCCD)
passages: 6 to 10
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| Treatment protocol |
For dDAVP treatment, cells were transferred to permeable supports (75mm, 0.4um pore, Corning #3419), and grown until confluency as documented transepithelial resistance measurements (Epithelial Volt ohmmeter; WPI). At that point, the complet medium was changed to serum- and hormone-free medium, and cells were maintained for 24 hr. Cells were incubated with the V2 receptor-selective vasopressin analog dDAVP (0.1 nM) or its vehicle (n=2) and harvested after 24 hr.
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| Growth protocol |
Cultured mouse mpkCCD (clone 11) cells were grown and maintained in complete medium, DMEM/F12 containing 2% dialyzed serum and supplements (5 μg/mL insulin, 50 nM dexamethasone, 1 nM triiodothyronine, 10 ng/mL epidermal growth factor, 60 nM sodium selenite, 5 μg/mL transferrin), as previously described (Yu MJ, Proc Natl Acad Sci U S A 106:2441-2446, 2009).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Shearing of chromatin was carried out by the manufacturer's protocol of truChIP kit (PN 520075, Covaris, Woburn, Massachusetts). All Covaris buffers were provided in kit. Confluent monolayers mpkCCD cells cultured on 100 mm permeable supports of transwell system were treated with dDAVP (0.1 nM) or vehicle for 24 h and then washed in ice-cold PBS followed by cross-linking in 1.11% formaldehyde in Covaris Fixing Buffer (5.5 ml) for 5 min at room temperature with gentle rocking. Cross-linking was terminated by the addition of 0.3 ml of 1X Covaris Quenching Buffer with rocking. The buffer solution was aspirated and ice-cold PBS was added to each plate. Cells were scraped, spun down (200xg, 5 min), and washed twice with ice-cold PBS. Cell pellets were resuspended in 1 ml of 1X Covaris Lysis Buffer for 10 min at 4°C with rocking. Nuclei were pelleted by spinning at 1,700xg for 5 min at 4°C. Nuclear pellets was resuspended in 1 ml of 1X Covaris Wash Buffer and incubated for 10 min at 4°C with rocking. Samples were spun at 1,700xg for 5 min at 4°C. Nuclear pellets were washed twice with 1 ml of Covaris Non-ionic Shearing Buffer and spun at 1,700xg for 5 min at 4°C. Nuclei were resuspended in 1 ml of Covaris Non-ionic Shearing Buffer. Two 20 ul aliquots of the resuspended pellets were removed prior to shearing to be used as subsequent unsheared controls for immunoblotting and agarose gel electrophoresis. The remaining samples were transferred to AFA tubes (TC12 x 12 mm). Samples were sheared using a Covaris S2 SonoLAB Single system for two minutes (Duty cycle: 5%; Intensity: 4; cycle: 200). Total nuclear protein concentration of each sample in mg/ml was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). To test shearing efficiency, a 20 ul aliquot from each sample was obtained after shearing. The aliquots from the unsheared sample and the sheared sample, were incubated with 100 ul 1X TE buffer, 10 ul 10% SDS, and RNAse A (10 ug/ul, Cell Signaling Technology, Danvers, Massachusetts) for 30 min at 37°C. After adding proteinase K (2 ug), the samples were incubated overnight at 65°C. The DNA was purified using DNA Clean & Concentrator columns (Catalog #: D4013, Zymo Research, Irvine, CA) and ran on an E-gel EX 2% agarose (Invitrogen, Carlsbad, CA). Target fragment sizes for chromatin immunoprecipitation were in the range of 150-700 bp in length with average fragment size of 300 bp. Immunoprecipitaiton was performed by the manufacturer's instruction (SimpleChIP protocol #9003, Cell Signaling Technology). 'Input' sample (10 ul) was obtained prior to immunoprecipitation. Chromatin Immunoprecipitation was performed using 30-90 ug of sheared chromatin in 500 uL of 1X Covaris Non-ionic Shearing Buffer with 10 μl anti-H3K27 acetylation antibodies. The mixtures were incubated overnight at 4°C with gentle rotation, followed by incubation with ChIP Grade Protein G Magnet Beads (Catalog #: 9006, Cell Signaling Technology) for 2 h at 4°C with rotation. The beads were washed a total of four times, including three low salt washes followed by one high salt wash for 5 min each at 4°C. Immunoprecipitated chromatin was eluted from the beads with 150 ul of 1X ChIP Elution Buffer for 30 mins at 65°C in a thermomixer. To reverse crosslinks, 6 ul of 5 M NaCl and 2 ul of 20 ug/ul Proteinase K were added to the chromatin followed by incubation at 65°C overnight. DNA was purified according to the manufacturer's instruction.
Sequencing libraries were prepared according to the manufacturer's instruction (NuGEN Ovation SP Ultralow library system kit). Adaptor-conjugated iibraries were amplified by PCR. The amplified libararies were measured. A fixed amount of the total measured DNA was used for sequencing in an Illumina Hiseq2000 instrument to obtain single read data. Libraries were sequenced using an Illumina HiSeq2000, obtaining 1- by 50-bp reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Align and transform the .fastq to .sam files using BWA program and mm10 mouse genome: bwa samse mm10.fa sample.sai sample.fastq > sample.sam bwa aln -t 8 mm10.fa sample.fastq > sample.sai.
Convert format from Sam to Bam and index the files using Samtools: Samtools view –bhS sample.sam > sample.bam, samtools sort sample.bam sample.sorted; samtools index sample.sorted.bam.
Create bedgraph files using macs2, macs2 callpeak -t sample_filename.sorted.bam -c input_filename.sorted.bam -f BAM -g mm -n output_filename –B –q 0.01 –SPMR
Create a peak txt file from a macs2 peak file
Create a bedGraph file from pile up file of macs2 output
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph and txt files
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| Submission date |
Feb 16, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Hyun Jun Jung |
| E-mail(s) |
hjung24@jhmi.edu
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| Organization name |
Johns Hopkins University School of Medicine
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| Department |
Medicine (Nephrology)
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| Street address |
720 Rutland Ave, Ross Research Building 1165B
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE95007 |
ChIP-Seq analysis of Histone H3 acetylation (K27) changes in response to vasopressin in mouse renal collecting duct mpkCCD cells |
| GSE95009 |
Protein kinase A-dependent signaling network revealed by multi-omic analysis |
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| Relations |
| BioSample |
SAMN06342946 |
| SRA |
SRX2568510 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2494231_exp2_h3k27ac_vehicle_peaks.txt.gz |
860.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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