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| Status |
Public on Oct 09, 2012 |
| Title |
pro-B, H3K27me3 ChIP seq |
| Sample type |
SRA |
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| Source name |
progenitor B
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| Organism |
Mus musculus |
| Characteristics |
cell type: Rag1 deficient pro-B
chip antibody: H3K27me3 (07-449, Millipore), lot# DAM1588246
chip antibody catalog #: DAM1588246
chip antibody catalog #: 07-449
chip antibody manufacturer: Millipore
genetic background: C57bl/6
genotype: Rag1-/-
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| Growth protocol |
Tcf(E2A)-deficient multipoint progenitors were derived from E2A-deficient fetal liver and grown in the presence of IL7, SCF and FLT3L on sub-confluent S17 stromal cells. B220 MACS enriched Rag1-deficient bone marrow cells were grown in Opti-MEM 10% FCS/ 2% PSG/ 50 μM β-mercaptoethanol in the presence of IL7 and SCF for 7 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq: Lysates were clarified from sonicated cells and protein-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (minus exo) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, ChIP DNA was PCR-amplified with Illumina primers with the respective indexes for 18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size- selected from a 8% polyacrylamide gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on a Genome Analyzer II (Illumina) or HiSeq 2000 (Illumina) following the manufacturer's protocols.
GRO-seq: GRO-Seq was performed as described (Core, L.J., Waterfall, J.J., and Lis, J.T., Science 322, 1845-1848 (2008)) with minor modifications. Briefly, nuclei from 10 million cells isolated by hypotonic lysis, and RNA polymerases were run-on for 5 minutes at 30°C in the presence of sarkosyl, BrUTP, ATP, GTP and limiting concentrations of CTP. Total RNA was purified with Trizol/isopropanol precipitiation, DNAse-treated (TURBO-DNase, Ambion), fragmented with fragmentation buffer (Ambion), and re-buffered by size exclusion chromatography. RNA fragments were 3' dephosphorylated with polynucleotide kinase (Enzymatics), and BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads, washed, eluted and EtOH-precipitated. Run-on RNA was then de-capped with tobacco acid pyrophosphatase (Epicentre), 5' phosphorylated with polynucleotide kinase (Enzymatics) and purified with Trizol LS/isopropanol precipitiation. Sequencing libraries were prepared by ligating a single-stranded, 3'-blocked, 5'-adenylated 3’ oligonucleotide with mutant (K227Q) truncated RNA ligase 2 (NEB) to the 3' end of the RNA fragments, followed by annealing a reverse transcription primer complementary to the 3' adapter to suppress adapter dimer formation, and ligating a hybrid 5' DNA-RNA oligonucleotide using RNA ligase I and then reverse-transcribing with SuperScript III reverse transcriptase (Invitrogen). The cDNA was purified with AMPure XL beads and PCR-amplified with primers bearing primer landing sites compatible with Illumina indexed sequencing. The library was size-selected on a PAGE gel to 60-110 bp insert size and sequenced on a HiSeq 2000 (Illumina).
Hi-C: The Hi-C experiment was performed similarly as described (Lieberman-Aiden, E., et al., Science 326, 289-293 (2009)). Libraries were subjected to paired-end sequencing on a HiSeq 2000 (Illumina).
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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 |
For ChIP-Seq and GRO-Seq samples (single end reads), reads were aligned to the mm9 genome using bowtie (v0.12.7), keeping only reads that mapped best to a single, unique location. Aligned read files were analyzed with HOMER (http://biowhat.ucsd.edu/homer/) to find peaks, calculate RPKM from the gene bodies of RefSeq genes, and perform other analyses in the study.
For Hi-C samples (paired end reads), read ends were aligned separately to the mm9 genome using bowtie (v0.12.7), only keeping read pairs if both ends mapped best to unique locations. Four additional filtering steps were applied to the mapped reads: 1) Duplicate read pairs aligning to the exact same positions were removed (possible PCR amplification artifacts). 2) Reads aligning within 2x the library size (estimated library insert size = 400) were removed (representing genomic background/religation events). 3) Read pairs where neither read was within 500 bp of a HindIII site were removed. (background ligation events) 4) Read pairs aligning to adjacent restriction enzyme sites representing circularization of a HindIII fragment were removed. Reads passing these filters are included in the Hi-C summary files. Read alignment filtering and other Hi-C processing including normalization was performed using HOMER (http://biowhat.ucsd.edu/homer/). Principle component analysis of normalized Hi-C interaction frequency matrices was performed using HOMER and R (www.r-project.org). Results for the first principle component are included as BEDGRAPH files. Positive principle component values correspond to regions that are generally transcriptionally active.
Genome_build: mm9
Supplementary_files_format_and_content: Processed files include BED files (ChIP-Seq peak positions), rpkm text files (GRO-Seq expression data), BEDGRAPH (principle component values along the genome for Hi-C), and Hi-C summary files (raw filtered read pairs from Hi-C data, columns: readName, chr1, 5' position1, strand1, chr2, 5' position2,strand2 [positions are 1-index based]). All genomic coordinates are relative to mm9 (NCBI 37) mouse assembly.
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| Submission date |
Aug 16, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
[email protected]
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| Organization name |
University of California, San Diego (UCSD)
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| Department |
Medicine
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| Street address |
9500 Gilman Dr. MC 0640
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE40173 |
Global changes in the nuclear positioning of chromatin domains and genomic interactions that orchestrate B cell fate |
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| Relations |
| SRA |
SRX178464 |
| BioSample |
SAMN01120333 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM987809_proB.H3K27me3.peaks.mm9.bed.gz |
86.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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