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| Status |
Public on Mar 25, 2021 |
| Title |
H3K9me3 ChIP-seq in iNeuron |
| Sample type |
SRA |
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| Source name |
iNeuron
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| Organism |
Homo sapiens |
| Characteristics |
cell type: neuron differentiated from iPSC
treatment: NT
chip antibody: 10 ug, Active Motif, Cat. No. 39765
harvested day: day7
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| Growth protocol |
For neuronal differentiation, 20-25 million iPSCs were plated on day 0 onto a 15 cm plate in N2 media composed of knockout DMEM/F12 media (Life Technologies Corporation, Cat. No. 12660012) with N2 supplement (Life Technologies Corporation, Cat. No. 17502048), 1x GlutaMAX (Thermofisher Scientific, Cat. No. 35050061), 1x MEM nonessential amino acids (NEAA) (Thermofisher Scientific, Cat. No. 11140050), 10 M ROCK inhibitor (Y-27632; Selleckchem, Cat. No. S1049), and 2 g/mL doxycycline (Clontech, Cat. No. 631311). N2 media was changed once a day for two more days. Day 3 cells were replated onto freshly-prepared poly-L-ornithine- (PLO; 0.1 mg/ml; Sigma, Cat. No. P3655-10MG) coated dishes as follows: Cells were washed with PBS, dissociated with accutase for 10 minutes at 37°C, washed and plated in i3Neuron Culture Media: BrainPhys media (STEMCELL Technologies, Cat. No. 05790) supplemented with 1x B27 Plus Supplement (ThermoFisher Scientific, Cat. No. A3582801), 10 ng/mL BDNF (PeproTech, Cat. No. 450-02), 10 ng/mL NT-3 (PeproTech, Cat. No. 450-03), 1 mg/mL mouse laminin (Sigma, Cat. No. L2020-1MG), and 2 g/mL doxycycline (Clontech, Cat. No. 631311).For 10 cm plates used in SAR-seq or CHIP-seq, 12-15 million neurons were plated. For 15 cm plates 30-45 million neurons were plated. For ibidi slides used in imaging experiments, 0.2 million neurons per well were plated. Unless otherwise noted, i3Neurons were fed on day 6 during a half media change and harvested on day 7. For i3Neurons cultured beyond 7 days, half media changes were conducted three times per week. In some experiments pre-differentiated i3Neurons were frozen on day 3 in 90% fetal bovine serum (Sigma Aldrich, Cat. No. ES-009-B) and 10% DMSO (Mediatech Inc., Cat. No. 25-950-CQC), and then thawed rapidly at 37°C, followed by removal of FBS/DMSO and plating in i3Neuron Culture Media. We did not detect any differences for experiments where day 3 neurons were thawed or plated immediately following differentiation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed in 1% formaldehyde. Cells were sonicated, lysates clarified, and protein-DNA complexes immunoprecipitated.
Libraries were quantified using with the KAPA Library Quantification Kit (Kapa Biosciences) and sequenced in a NextSeq 550 system (Illumina, 75 bp single end reads). libraries were prepared and sequenced following standard Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Data processing |
SAR-seq, END-seq, ChIP-seq, SEAL, and ATAC-seq reads were aligned to the reference genome (hg19 for human i3Neuron and iMuscle, mm10 for mouse pre B cells or rn6 for rat primary neurons) using bowtie (v1.1.2) with parameters -n 3 -l 50 -k 1 for END-seq and -n 2 -l 50 -m 1 for the rest. RNA-seq reads were aligned by STAR (v2.7.6a). Functions “view” and “sort” of samtools (v 1.6) were used to convert and sort the aligned sam files to sorted bam files. Bam files were further converted to bed files by the bedtools bamToBed command. Mitochondrial reads were removed in SAR-seq for intensity comparisons.
We used MACS (v1.4.3) to call SAR-seq, XRCC1 ChIP-seq and ATAC-seq peaks. SAR-seq XRCC1, and CTCF ChIP-seq peaks with >10 fold-enrichment over background were kept. Peaks of hisone modification determined by ChIP-seq peaks were called by SICER with default parameters. Peaks within blacklisted regions (https://sites.google.com/site/anshulkundaje/projects/blacklists) were filtered. As peaks of ddN S1-END-seq are always clustered, subpeaks were called by the PeakSplitter tool of PeakAnalyzer with parameters -c 15 -v 0.6.
BedGraph files were generated by bedtools genomecov, normalized by reads per million (RPM) and then converted to bigWig files using bedGraphToBigWig from UCSC pre-compiled utilities for visualization at UCSC genome browser
Genome_build: hg19 for human, mm10 for mourse, rn6 for rat.
Supplementary_files_format_and_content: bigwig, bed
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| Submission date |
Feb 22, 2021 |
| Last update date |
Mar 25, 2021 |
| Contact name |
Wei Wu |
| Organization name |
Center for Excellence in Molecular Cell Science
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| Department |
Center for Excellence in Molecular Cell Science
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| Street address |
320 yueyang road
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| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE167253 |
Neuronal Enhancers are Hotspots for DNA Single-Strand Break Repair [ChIP-seq] |
| GSE167259 |
Neuronal Enhancers are Hotspots for DNA Single-Strand Break Repair |
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| Relations |
| BioSample |
SAMN18027991 |
| SRA |
SRX10151296 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5100372_H3K9me3_ChIPseq_iNeuron.bw |
226.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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