 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 07, 2016 |
| Title |
ChIP-seq_H3K4me3_WT_rods_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Rods from WT mouse retina
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL6J/129
genotype/variation: LMOPC1-Cre; R26-LSL-CAG-Sun1-GFP-myc
age: 8 to 11 weeks
Sex: male
tissue: retina
cell type: WT rod photoreceptors
chip antibody: rabbit anti-H3K4me3 (Abcam ab8580)
|
| Growth protocol |
Adult mice were housed in our animal facility with 12 h light/dark cycles and food ab libitum. Animals were used for analysis in accordance with protocols approved by the Institutional Animal Care and Use Committee
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The retina was rapidly dissected from 1-2 adult mice on ice, and the INTACT method was used to affinity purify GFP+ nuclei. Nucleosomes for native ChIP-seq were prepared as previously described [Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Briefly, 1-2 million bead-bound nuclei were digested with 0.025 units/uL micrococcal nuclease (Worthington LS004798) in 500uL of 15mM HEPES pH 7, 1mM KCl, 2mM MgCl2, 2mM CaCl2, 340mM sucrose, 0.15mM spermine, 0.5mM spermidine, and 5mM sodium butyrate at 37°C for 15 minutes. The reaction was terminated by the addition of EGTA to 2mM final concentration. Nucleosomes were extracted for 30 minutes on ice with 200uL 15mM HEPES pH7, 200mM NaCl, 25mM KCl, 2mM MgCl2, 1mM EGTA, 340mM sucrose, 0.15mM spermidine, 0.15mM spermine, and 5mM sodium butyrate. A second 30 minute extraction was performed with the same buffer except the salt concentration was raised to 400mM NaCl. The extracts were combined and dialyzed overnight against 15mM HEPES pH7, 25mM KCl, 1mM β-mercaptoethanol, 1mM PMSF, and 5mM sodium butyrate using a 10K cut-off Slide-a-Lyzer dialysis device (Thermo Scientific 88401).
Native ChIP and library construction were combined [Garber M. et al. A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals. Mol. Cell 47, 810-822 (2012); Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Nucleosomes prepared from 0.5-1 million nuclei were incubated with 1ug antibody and 25uL Protein G Dynabeads. The following antibodies were used: rabbit anti-H3K27me3 (Millipore 07-449), rabbit anti-H3K27ac (Abcam ab4729), rabbit anti-H3K4me3 (Abcam ab8580), and rabbit anti-H3K4me1 (Abcam ab8895). ChIP-enriched and input DNA was end-repaired, linker adapted, and sequenced on an Illumina HiSeq 2500 for 50 cycles.
RNA-seq: Mice retinas were rapidly dissected on ice, and either the INTACT method or flow cytometry was used to purify GFP+ nuclei. Isolated nuclei were resuspended in Buffer RLT for RNA purification using the RNeasy Micro kit (Qiagen 74004) with on-column DNase digestion following the standard kit protocol. RNA quality was measured by an Agilent Bioanalyzer.
RNA-seq: For preparation of nuclei from the whole retina, the retina was rapidly dissected from 1 adult mouse on ice, homogenized, and nuclei were pelleted by centrifugation. Pelleted nuclei were resuspended in Buffer RLT for RNA purification using the RNeasy Micro kit (Qiagen 74004) with on-column DNase digestion following the standard kit protocol. RNA quality was measured by an Agilent Bioanalyzer.
Bisulfite-Seq (MethylC-seq): Mice retinas were rapidly dissected on ice, and either the INTACT method or flow cytometry was used to purify GFP+ nuclei. Isolated nuclei were resuspended in PBS for DNA purification using the DNeasy Blood and Tissue kit (Qiagen 69504) following the standard kit protocol.
ATAC-seq: Mice retinas were rapidly dissected on ice, and the INTACT method was used to purify GFP+ nuclei.
ChIP-seq: Mice retinas were rapidly dissected on ice, and the INTACT method was used to purify GFP+ nuclei. Nucleosomes for native ChIP-seq were prepared as previously described [Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Briefly, 1-2 million bead-bound nuclei were digested with 0.025 units/uL micrococcal nuclease (Worthington LS004798) in 500uL of 15mM HEPES pH 7, 1mM KCl, 2mM MgCl2, 2mM CaCl2, 340mM sucrose, 0.15mM spermine, 0.5mM spermidine, and 5mM sodium butyrate at 37°C for 15 minutes. The reaction was terminated by the addition of EGTA to 2mM final concentration. Nucleosomes were extracted for 30 minutes on ice with 200uL 15mM HEPES pH7, 200mM NaCl, 25mM KCl, 2mM MgCl2, 1mM EGTA, 340mM sucrose, 0.15mM spermidine, 0.15mM spermine, and 5mM sodium butyrate. A second 30 minute extraction was performed with the same buffer except the salt concentration was raised to 400mM NaCl. The extracts were combined and dialyzed overnight against 15mM HEPES pH7, 25mM KCl, 1mM β-mercaptoethanol, 1mM PMSF, and 5mM sodium butyrate using a 10K cut-off Slide-a-Lyzer dialysis device (Thermo Scientific 88401).
RNA-seq: RNA (2-50ng) was converted to cDNA and amplified using Nugen Ovation RNA-seq System V2 (Nugen 7102). All RNA samples received a 1:10,000 dilution of ERCC RNA (Life Technologies 4456740). Amplified cDNA was fragmented, end-repaired, linker adapted, and sequenced for 50 cycles on an Illumina HiSeq 2500 instrument.
Bisulfite-Seq (MethylC-seq): MethylC-seq libraries were constructed as previously described (Mo et al., 2015). Libraries were sequenced on an Illumina HiSeq 2000 up to 101 cycles.
OTHER: ATAC-seq: Approximately 50,000 bead-bound nuclei were transposed in a 50uL volume of 1X TD buffer and 2.5uL Tn5 transposase (Illumina FC-121-1030) for 30 minutes at 37°C, as previously described (Buenrostro et al., 2013), with the modification that fragmented genomic DNA was recovered using Buffer QG (Qiagen 28604). Transposed genomic DNA was amplified by five cycles of quantitative PCR. 10% of the PCR was subjected to an additional 20 cycles of SYBR green-based qPCR while the remainder of the sample was left on ice. Analysis of the qPCR data allowed a rough estimate of the number of additional cycles needed to generate product at 25% saturation. Typically, four to seven additional PCR cycles were added to the initial set of five cycles. Amplified DNA was purified on AMPure XP beads, analyzed on an Agilent Bioanalyzer, and sequenced (paired-end) on an Illumina HiSeq 2500 for 101 cycles.
ChIP-seq: Native ChIP and library construction were combined [Garber M. et al. A high-throughput chromatin immunoprecipitation approach reveals principles of dynamic gene regulation in mammals. Mol. Cell 47, 810-822 (2012); Henry G.L. et al. Cell type-specific genomics of Drosophila neurons. Nucleic Acids Res. 40, 9691-9704 (2012)]. Nucleosomes prepared from 0.5-1 million nuclei were incubated with 1ug antibody and 25uL Protein G Dynabeads. The following antibodies were used: rabbit anti-H3K27me3 (Millipore 07-449), rabbit anti-H3K27ac (Abcam ab4729), rabbit anti-H3K4me3 (Abcam ab8580), and rabbit anti-H3K4me1 (Abcam ab8895). ChIP-enriched and input DNA was end-repaired, linker adapted, and sequenced on an Illumina HiSeq 2500 for 50 cycles.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
processed data files: ChIP-seq_H3K4me3_WT_rods_SICER_peaks.gz
|
| Data processing |
Data was mapped to the Mus musculus reference genome (mm10).
Analysis for RNA-seq, MethylC-seq, ATAC-seq, and ChIP-seq datasets were performed as described in Mo et al., 2015 (in submission)
RNA-seq: Five base pairs were trimmed from the 5'-end of all reads to remove possible adaptor contamination (seqtk trimfq -b 5)
RNA-seq: Trimmed reads were aligned to the mm10/ERCC92 combined index using TOPHAT v1.4.0 (--GTF --no-novel-juncs) guided by protein coding gene annotation from iGenomes
RNA-seq: Gene expression levels were estimated using RSEM v1.1.20 (--fragment-length-mean 250 --fragment-length-sd 50)
RNA-seq: Visualization tracks were generated by counting genome-wide coverage of reads (BEDTOOLS v2.15.0 genomeCoverageBed -split -bg), scaling to 10M total alignments (custom perl script), and converting to bigWig format (UCSC wigToBigWig).
MethylC-seq: MethylC-seq reads were processed as previously described using the methylpy pipeline (https://bitbucket.org/schultzmattd/methylpy/) for alignment and mC calling
MethylC-seq: DMRs were identified using DSS [Feng H. et al. A Bayesian hierarchical model to detect differentially methylated loci from single nucleotide resolution sequencing data. Nucleic Acids Res. 42, e69 (2014)].
MethylC-seq: UMRs and LMRs were identified using MethylSeekR [Burger et al. Identification of active regulatory regions from DNA methylation data. Nucleic Acids Res. 41, e155 (2013)] using m (methylation) = 0.5 and 5% FDR.
ATAC-seq: Adapter sequences were trimmed from ATAC reads (cutadapt v1.3 -a CTGTCTCTTATACACATCT -q 30 --minimum-length 36 --paired-output)
ATAC-seq: Trimmed reads were aligned to the mm10 genome (BOWTIE2 v2.1.0 -t -X2000 --no-mixed --no-discordant)
ATAC-seq: Redundant reads were removed (picard MarkDuplicates).
ATAC-seq: Peaks were called with MACS2 [Zhang Y. et al. Model-based analysis of ChIP-Seq. Genome Biol. 9, R137 (2008)] (macs2 2.1.0.20140616 callpeak -p 0.00001 --call-summits --nomodel --shift -50 --extsize 100) using sub-nucleosomal (<100bp) fragments. File format is MACS2 narrowPeak.
ATAC-seq: Visualization tracks were generated by counting genome-wide coverage of all fragments (BEDTOOLS v2.15.0 genomeCoverageBed -bg), scaling to 10M total alignments (custom perl script), and converting to bigWig format (UCSC wigToBigWig).
ChIP-seq: Reads were aligned to the mm10 genome, keeping only uniquely aligning reads (BOWTIE v0.12.7 -m 1)
ChIP-seq: Visualization tracks were generated by counting genome-wide coverage of reads (BEDTOOLS v2.15.0 genomeCoverageBed -bg), scaling to 10M total alignments (custom perl script), and converting to bigWig format (UCSC wigToBigWig).
ChIP-seq: Peaks were called with SICER_V1.1 (redundancy threshold = 1; fragment size=150; W=200, G=200 for H3K27ac, H3K4me1, and H3K4me3; W=200, G=1000 for H3K27me3). Overlapping peaks from the 2 replicates were merged (BEDTOOLS merge) to generate 1 set of peaks for each histone modification.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig tracks for visualization of RNA-seq, ATAC-seq, and ChIP-seq datasets
tab delimited text files of gene expression levels estimated using RSEM v1.1.20
tab delimited text files of methylcytosine calls; columns in allc files are: column 1 - chromosome; column 2 - position; column 3 - strand; column 4 - class; column 5 - mC reads; column 6 - total reads; column 7 - methylated (Boolean value indicating the result of statistical test for methylated cytosines)
tab delimited text files of DMRs; columns in DMRs files are: column 1 - chromosome; column 2 - start; column 3 - end; column 4 - category
tab delimited text files of UMRs and LMRs; columns in UMRs/LMRs files are: column 1 - chromosome; column 2 - start; column 3 - end; column 4 - UMR or LMR
tab delimited text files of ATAC-seq peaks; columns in peak files are: column 1 - chromosome; column 2 - start; column 3 - end
tab delimited text files of ChIP-seq peaks; columns in peak files are: column 1 - chromosome; column 2 - start; column 3 - end
|
| |
|
| Submission date |
Aug 31, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Alisa Mo |
| E-mail(s) |
amo4@jhmi.edu
|
| Phone |
410 955 4679
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Molecular Biology and Genetics
|
| Lab |
Jeremy Nathans
|
| Street address |
725 N. Wolfe St. PCTB805
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE72550 |
Epigenomic Landscapes of Retinal Rods and Cones |
|
| Relations |
| BioSample |
SAMN04021774 |
| SRA |
SRX1175790 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1865028_ChIP-seq_H3K4me3_WT_rods_rep2_scaled10M.bw |
86.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
