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Sample GSM6428036 Query DataSets for GSM6428036
Status Public on Dec 01, 2022
Title iNeuron_WT_controlfor_sgTDG_PBseq_rep2
Sample type SRA
Source name iNeuron
Organism Homo sapiens
Characteristics harvested day: day7
genotype: WT
treatment: NT
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.
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 ( 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).
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina MiSeq
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
Submission date Aug 02, 2022
Last update date Dec 03, 2022
Contact name Wei Wu
Organization name Center for Excellence in Molecular Cell Science
Department Center for Excellence in Molecular Cell Science
Street address 320 yueyang road
City Shanghai
State/province Shanghai
ZIP/Postal code 200031
Country China
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
BioSample SAMN30102023
SRA SRX16769824

Supplementary file Size Download File type/resource
GSM6428036_iNeuron_WT_controlfor_sgTDG_PBseq_rep2.methratio.txt.gz 8.5 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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