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| Status |
Public on Dec 05, 2016 |
| Title |
ATAC_seq_day6 |
| Sample type |
SRA |
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| Source name |
Postmitotic motor neurons day 6
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| Organism |
Mus musculus |
| Characteristics |
cell type: Ainv15 ES cell-derived spinal motor neurons
time: day 6
growth protocol: iCre; RA, SAG on day 2; DAPT on days 4,5
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| Growth protocol |
Motor neuron differentiation of ESCs was performed as described previously (Wichterle et al., 2002) with some modification. Briefly, ESCs were trypsinized and seeded at 5 x 10^4 cells per mL (day 0). 1 uM RA and 0.25 uM SAG were added to the medium on day 2. 5 uM DAPT was added on days 4 and 5. For inducible Ngn2 and NIL lines, 3 ug/ml of doxycycline (Dox) was added on day 3 and day 2, respectively.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq and ChIP-exo experiments, cells were lysed, and chromatin pellets were isolated and then solubilized and fragmented by sonication. Fragmented chromatin was then subject to immunoprecipitation using antibodies against protein of interest. RNA was extracted using TRIzol reagent (Thermo 15596026). Illumina Ribo-Zero rRNA Removal Kit (MRZH116) was used with DNase-treated total RNA sample to remove ribosomal RNA.
ChIP-seq libraries were prepared according to standard Illumina's instructions for HiSeq2000. Briefly, DNA was end-repaired, and ligated to sequencing adaptors, then PCR amplified and gel purified. The purified DNA libraries were sequenced following the manufacturer's protocols. For ChIP-exo libraries, while immunoprecipitates were still on the beads, samples were end-repaired, and ligated to a first sequencing adaptor, and subjected to lambda exonuclease digestion. Samples were then eluted from the beads and converted to double-stranded DNA by primer annealing and extension. A second sequencing adaptor was then ligated to exonuclease treated ends, then DNA was PCR amplified, gel purified, and sequenced. ATAC-seq libraries were prepared as described previously (Buenrostro et al., 2013). Briefly, cells were lysed and spun down. The pellet was resuspended in 50 ul of the transposase reaction mix containing 2.5 μL Tn5 transposase and sequencing adaptors (Illumina DNA Library Preparation Kit, FC-121-1030), and incubated for 30 min at 37 °C. The purified DNA was PCR amplified and sequenced. RNA-seq libraries were prepared for sequencing using standard Illumina protocols. DNase-seq libraries were prepared as described previously (He et al., 2014). Briefly, cells were lysed and spun down. The pellet was resuspended in 500 uL pre-warmed 15 mM Tris buffer A (pH8.0) containing 15 mM NaCl, 60 mM KCl, and 0.5 mM spermidine, and then add different amounts (0, 25, 50, and 75U) of DNase I (Roche, 04716728001) for 5 min at 37C. The reactions were terminated by 500 uL Stop buffer containing 0.5 mM spermidine and 1 ug of RNase, and incubated at 55C for 15 min. The purified DNA was sequenced by following standard Illumina protocols.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
library strategy: ATAC-seq
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| Data processing |
ChIP-seq sequencing reads were aligned to the mm9 genome assembly using Bowtie (version 0.12.7).
Peaks were called using the Genome Positioning System (GPS) and the GeneTrack to identify regions enriched by TFs over background.
For ChIP-seq H3K27ac data sets, peaks were called using Model-based Analysis of ChIP-Seq (MACS).
For ATAC-seq data, peaks were called using Homer software (v4.7.2).
For DNase-seq data, peaks were called using the GeneTrack peak finding algorithms (Albert et al., 2008).
RNA-seq sequencing reads were aligned to the mouse genome using RNA-seq alignment algorithm in the STAR (Spliced Transcripts Alignment to a Reference) software.
To assemble transcripts and estimate the relative abundances of these transcripts
The FPKM (Fragments Per Kilobase of exon per Million fragments mapped) was calculated per individual gene.
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files include: FPKM values for RNA-seq samples; GPS peak calls and GeneTrack peak calls for TF ChIP-exo and ChIP-seq data. BED format files include: MACS peak calls for histone modification; Homer peak calls for ATAC-seq.
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| Submission date |
Mar 23, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hynek Wichterle |
| Organization name |
Columbia University Medical Center
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| Department |
Departments of Pathology and Cell Biology, Neurology, and Neuroscience
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| Street address |
630 West 168th Street
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE79561 |
Dynamic enhancer landscape in postmitotic motor neurons |
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| Relations |
| BioSample |
SAMN04577761 |
| SRA |
SRX1659054 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2098391_d6.mm9.100.peaks.bed.gz |
918.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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