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| Status |
Public on May 03, 2019 |
| Title |
HuKidney_WT_pCREB_rep2 |
| Sample type |
SRA |
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| Source name |
adult human kidney cortex
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| Organism |
Homo sapiens |
| Characteristics |
tissue: kidney cortex
Sex: female
antibody: pCREB (EMD Millipore, 06-519, lot# 3108493)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed as described previously (Meyer MB, JBC. 2017). Briefly, samples were subjected to immuno-precipitation using either a control IgG antibody or the indicated experimental antibody (VDR, pCREB, H3K4me1, or H3K27Ac). The isolated DNA (or Input DNA acquired prior to precipitation) was then validated by quantitative real time PCR (qPCR) and further prepared for ChIP-seq analysis.
ChIP-seq libraries were prepared using the NEBNext DNA sample prep kit (NEB, #E6000L) with the Bioo NEXTflex ChIP-seq Barcodes (Bioo Scientific, Austin, TX, #514122) according to manufacturer’s protocols, with few exceptions. During the NEBNext prep, the Illumina adapters were replaced with the Bioo Scientific Barcoded adapters according to Bioo protocols. ChIP-DNA ligated libraries were cleaned up with Agencourt AMPureXP Magnetic Beads (Beckman-Coulter, #A63881). A pre-size selection PCR was performed using Phusion polymerase, NEXTflex Primer Mix and purified ligation product for 4 cycles of PCR according to the Bioo Protocol. Libraries were size selected using Invitrogen E-gels to a size of 400-500bp. Samples were then PCR amplified for 14 cycles using Phusion polymerase, NEXTflex Primer mix and the size selected DNA as per Bioo protocol, followed by Agencourt bead clean up. Libraries were validated for integrity using the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Clusters were formed and sequenced on the Illumina HiSeq2000 sequencers by the University of Wisconsin-Madison DNA Sequencing Facility in the University of Wisconsin-Madison Biotechnology Center. DNA clusters were generated using a cBot Single Read Cluster Generation kit (ver. 3) on an Illumina cBot (Illumina, Carlsbad, CA) according to the manufacturer's instructions, to obtain an average of 1.5×108 clusters for each lane on a flowcell. All sequencing runs for 100mers were performed on an Illumina HiSeq2000 using the Illumina Sequencing kit (ver. 3). Fluorescent images were analyzed using the CASAVA 1.8.2 (Illumina Carlsbad, CA) to obtain FASTQ formatted sequence data. Twelve barcoded libraries were run per lane and this was repeated over 4 lanes. After which, the raw FASTQ for each sample was concatenated from the 4 lanes prior to mapping to create a single sample. The ChIP samples were repeated in biological triplicate. Sequences were mapped to the human genome (hg19) using BOWTIE (--best –m 1) to yield unique alignments.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
ChIP-DNA
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| Data processing |
ChIP-seq runs were all 100bp. Barcoded samples were run over 4 lanes total and the fastq data were concatenated prior to Bowtie2 mapping. 12 barcoded samples were run in each lane, over 4 lanes total. Barcodes were decoded by Illumina HiSeq2000 software automatically. All samples were mapped from fastq files using Bowtie2 to hg19 [UCSC human genome build 19]. Triplicate lanes were analyzed separately for reproducibility and normalization of the peak calls.
Peaks were called by using MACS2. Peak finding for MACS2 was standard narrowPeak for TFs and broadPeak [--broad] for histones. Reported as bed files for narrowPeak and broadPeak files.
Peaks were called by using HOMER [http://biowhat.ucsd.edu/homer/]. HOMER also output the bedGraph files of read density for UCSC genome display. HOMER settings for peak finding were all standard settings.
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files are suitable to display in the UCSC genome browser. *peaks.bed files were produced by HOMER. *narrowPeak files were produced by MACS2
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| Submission date |
Apr 10, 2019 |
| Last update date |
May 05, 2019 |
| Contact name |
Mark B Meyer |
| E-mail(s) |
markmeyer@wisc.edu
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| Phone |
608-890-0857
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| Organization name |
University of Wisconsin-Madison
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| Department |
Nutritional Sciences
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| Lab |
Meyer Lab
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| Street address |
1415 Linden Dr.
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| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE129585 |
ChIP-seq analysis of adult human kidney cortex |
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| Relations |
| BioSample |
SAMN11388431 |
| SRA |
SRX5661983 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3716709_HuKidney_WT_pCREB_Veh_rep2.peaks.bed.gz |
913.9 Kb |
(ftp)(http) |
BED |
| GSM3716709_HuKidney_WT_pCREB_Veh_rep2.tagdirnew.ucsc.bedGraph.gz |
65.0 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM3716709_HuKidney_WT_pCREB_Veh_rep2_MACS_norm_peaks.narrowPeak.gz |
2.5 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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