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| Status |
Public on Jan 03, 2019 |
| Title |
NSH-B_K27M-Tag_input_DNA |
| Sample type |
SRA |
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| Source name |
Mouse e15.5 hindbrain neurosphere
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| Organism |
Mus musculus |
| Characteristics |
experiment id: 2
full genotype: Nestin-cre; H3f3aLSL-K27M-Tag/+
gender: female
growth protocol: Grown under neural precursor cell conditions
chip antibody: none
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tumors were snap frozen and ground to a powder before fixation. Single cell suspensions were generated from cultured cells using Accutase before fixation. Samples were fixed for 5 minutes with 1% paraformaldehyde in PBS (from a frozen stock) at room temperature. Bare nuclei were suspended in shearing buffer + protease inhibitors (10 Tris HCL pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS) and sheared on a Covaris M220 ultrasonicator (Microtube, 75W, 5% duty cycle, 200 cycles per burst, 5 minutes or Millitube, 75W, 10% duty cycle, 200 cycles per burst, 15 minutes). Sonication size was verified on an Agilent Bioanalyzer using a High Sensitivity DNA Assay. For ChIP-seq, sheared chromatin from drosophila melanogaster S2 cells was added to the mammalian chromatin prior to ChIP at a set ratio for each histone mark to allow for sample normalization. 400 ng of chromatin was used per ChIP reaction and for ChIP-seq, multiple ChIPs were performed and pooled as needed to generate enough chromatin for library construction. 10-20 ng of pre-IP chromatin containing spike in was kept as an input control. ChIP reactions were performed using a modified Upstate Biotechnology protocol. Briefly, sheared chromatin was diluted 1:10 with dilution buffer + protease inhibitors (21 Tris HCL pH 8.0, 1 mM EDTA pH 8.0, 167 mM NaCl, 1.1% Triton X-100, 0.1% SDS), precleared with 30 ul Fast Flow Protein A sepharose beads (GE Healthcare) and 50 μg bovine serum albumin (BSA) by rotating in a siliconized eppendorf tube at 4 C for 1-2 hours. The precleared chromatin was transferred to a tube containing the antibody of interest (see below), 30 ul Protein A sepharose beads and 50 ug BSA and rotated overnight at 4 C. The bead bound chromatin was washed once each with low salt buffer (20 mM Tris HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS), high salt buffer (20 mM Tris HCl pH 8.0, 2 mM EDTA pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS), LiCl buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 1% Nonidet P-40, 1% Deoxycholate) and twice with TE (10 mM Tris HCl pH 8.0, 1 mM EDTA pH 8.0). The chromatin was eluted off the beads with 0.1 M NaHCO3 /1% SDS by rotating at room temperature for 30 minutes, then the supernatant was transferred to a tube with 200nM NaCl and cross links were reversed by incubating overnight at 65 C (input controls were processed along with the ChIP samples from this point on). 10ug Proteinase K was added and the tube was incubated for 2 hours at 37 C, then the DNA was cleaned up using a QiaQuick PCR Purification Kit (Qiagen) and eluted in 50 μl 10 mM Tris HCl pH 8.5. Antibodies used were (μl antibody per IP shown): H3K27me3 (Cell Signaling 9733, lot 8; 4 μl), H3K27ac (Cell Signaling 8173, lot 1; 4 μl) and H3K4me3 (Cell Signaling 9751, lot 8; 5 μl).
Libraries were prepared from 5-10 ng of DNA using the NEBNext ChIP-Seq Library Prep Reagent Set for Illumina with NEBNext Q5 Hot Start HiFi PCR Master Mix according to the manufacturer’s instructions (New England Biolabs) with the following modifications: a second 1:1 Ampure cleanup was added after adaptor ligation. The Ampure size selection step prior to PCR was eliminated. Completed libraries were analyzed for insert size distribution on a 2100 BioAnalyzer High Sensitivity kit (Agilent) or Caliper LabChip GX DNA High Sensitivity Reagent Kit (Perkin Elmer). Libraries were quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies), Kapa Library Quantification kit (Kapa Biosystems) or low pass sequencing on a MiSeq Nano v2 run (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
We used BWA (version 0.7.12, default parameter) to align the reads to the mouse mm9 genome
marked duplicated reads with Picard with only nonduplicated reads kept by samtools
To control the quality of the data and estimate the fragment size, the nonduplicated version of SPP (version 1.11) was used to draw cross-correlation with support of R (version 2.14.0)
peaks were called using MACS2 or Sicer V1.1
bam was converted to BigWig using bedtools (v2.25.0) and UCSC tools (20150811). Read coverage was scaled according to spike-in normalization factor (Orlando et al., 2014)
Genome_build: mm9
Supplementary_files_format_and_content: bed files containing locations of defined peaks
Supplementary_files_format_and_content: bigWig files containing normalized read coverage
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| Submission date |
Dec 20, 2017 |
| Last update date |
Jan 03, 2019 |
| Contact name |
Hongjian Jin |
| E-mail(s) |
hongjian.jin@STJUDE.ORG
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| Organization name |
St Jude Children's Research Hospital
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| Department |
Center for Applied Bioinformatics
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| Street address |
262 Danny Thomas Place
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| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38015 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE108344 |
Spontaneous DIPG Modeling Reveals Novel H3.3 K27M-Mediated Oncogenic Mechanisms Acting Through Epigenetic Effects [ChIP-seq] |
| GSE108364 |
Spontaneous DIPG Modeling Reveals Novel H3.3 K27M-Mediated Oncogenic Mechanisms Acting Through Epigenetic Effects |
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| Relations |
| BioSample |
SAMN08214545 |
| SRA |
SRX3490295 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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