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| Status |
Public on Dec 01, 2022 |
| Title |
iNeuron_sgTDG_PBseq_rep3 |
| Sample type |
SRA |
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| Source name |
iNeuron
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| Organism |
Homo sapiens |
| Characteristics |
harvested day: day7
genotype: TDG CRISPRi
treatment: NT
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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 medium (knockout Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium; Life Technologies Corporation, cat. no. 12660012) with N2 supplement (Life Technologies, cat. no. 17502048), 1× GlutaMAX (Thermofisher Scientific, cat. no. 35050061), 1× MEM nonessential amino acids (NEAA) (Thermofisher Scientific, cat. no. 11140050), 10 μM ROCK inhibitor (Y-27632; Selleckchem, cat. no. S1049), and 2 μg/ml doxycycline (Clontech, cat. no. 631311). N2 medium was changed once a day for two more days. On day 3, cells were replated onto freshly prepared dishes coated with freshly prepared poly-L-ornithine (PLO; 0.1 mg/ml; Sigma, cat. no. P3655-10MG). Pre-neuron cells were cultured in i3Neuron Culture Media: BrainPhys media (Stemcell Technologies, cat. no. 05790) supplemented with 1× B27 Plus Supplement (ThermoFisher Scientific, cat. no. A3582801), 10 ng ml−1 BDNF (PeproTech, cat. no. 450-02), 10 ng ml−1 NT-3 (PeproTech, cat. no. 450-03), 1 μg ml−1 mouse laminin (Sigma, cat. no. L2020-1MG), and 2 μg ml−1 doxycycline (Clontech, cat. no. 631311). i3Neurons were then fed by half media change on day 6 and then, collected on day 7. For iMacrophage transdifferentiation, 10 million C10 cells were induced to reprogram by the addition of 100 nM of β-estradiol (Sigma-Aldrich; Cat. #E8875) and grown with 10 ng/ml of IL-3 (Peprotech; Cat. #213-13), CSF-1 (Peprotech; Cat. #315-02) and 350 nM Ascorbic acid (Sigma-Aldrich; Cat. # A8960) and then collected on day 2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from day 7 i3Neurons and day 2 C/EBPα-induced Macrophages using DNeasy® Blood & Tissue (QIAGEN; Cat. #69504) following manufacturer’s instructions.
100 ng genomic DNA was used in a 50-μl reaction containing 600 mM sodium acetate solution (3M, pH = 4.3) (Quality Biological, 351-309-101) and 1 M pyridine borane (Alfa Aesar, L13178-09), and incubated for 16 h at 37°C in a Eppendorf ThermoMixer shaking at 850 rpm. The product was purified by QIAquick Nucleotide Removal Kit (QIAGEN; Cat. # 28306). 4 primer pairs, covering 26 CpGs within neuronal enhancer regions were designed using BiSearch Web (http://bisearch.enzim.hu). Amplicons were between 200-250 base pairs. We also designed 3 primer pairs, which cover 19 CpGs within macrophage enhancer regions. The PCR amplicons were generated using the PyroMark PCR kit (QIAGEN; Cat. # 978703) and quantified using Qubit assays (Invitrogen). PCR amplicons were pooled, then library preparation steps were performed as described (PMID: 33767446). The final libraries were quantified using the KAPA library quantification kit for Illumina (KAPA Biosystems) and sequenced on Illumina Miseq (150 bp, paired end).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
SAR-seq, END-seq and SEAL reads were aligned to the reference genome (hg19 for human i3Neuron and mm10 for mouse macrophage cells 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). PB-seq was mapped by BSMAP (v2.90) with parameters -p 10 -w 2 -v 0 -q 30. Functions “view” and “sort” of samtools (v1.11) 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 (v2.29.2) bamToBed command. Mitochondrial reads were removed in SAR-seq for intensity comparisons.
Assembly: hg19 for human, mm10 for mourse.
Library strategy: PB-seq
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| Submission date |
Aug 02, 2022 |
| Last update date |
Dec 03, 2022 |
| 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 |
GPL15520 |
| Series (2) |
| GSE210313 |
Active DNA demethylation promotes cell fate specification and the DNA damage response [PB-Seq] |
| GSE210317 |
Active DNA demethylation promotes cell fate specification and the DNA damage response |
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| Relations |
| BioSample |
SAMN30102025 |
| SRA |
SRX16769821 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6428034_iNeuron_sgTDG_PBseq_rep3.methratio.txt.gz |
8.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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