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| Status |
Public on Dec 01, 2022 |
| Title |
iNeuron_ddA_SAR-seq |
| Sample type |
SRA |
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| Source name |
iNeuron
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| Organism |
Homo sapiens |
| Characteristics |
genotype: WT
treatment: ddA, 18 hours prior to cell harvesting
dose: 40 µM
harvested day: day7
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| Treatment protocol |
Neurons and iMacrophage cells were incubated with 20 uM EdU for 18 hours, unless otherwise noted.
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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 the 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 |
15 million i3Neuron or iMacrophages were incubated with 20 μM EdU for 18 hr. For i3Neurons, cells were harvested and washed with PBS and incubated with accutase for 5-10 minutes. Cells were then collected using a cell scrapper (Corning), pelleted at 200xg for 5 minutes and resuspended in cold 0.1% BSA in PBS. For iMacrophages, cells were washed, trypsinized and resuspended in cold 0.1% BSA in PBS. Cold methanol was then added dropwise into all samples during slow vortexing to 80% final concentration. Samples were stored at -20C until processed.
Cells were permeabilized on ice for 10 min and resuspended in 1 mL of Click-iT reaction solution for 2 hrs shaking at room temperature. Cells were then lysed with Proteinase K overnight at 37C. DNA was extracted with UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) (Invitrogen) according to manufacturer’s instructions. DNA was sonicated to 150-200 bp fragments using a Covaris S220 sonicator. Biotin-EdU labeled DNA fragments were pulled down using MyOne Streptavidin C1 Beads (ThermoFisher #650-01) and incubated at 24C shaking in a ThermoMixer C at 800 rpm for 30 min. Dynabeads were washed 3x in W&B (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 M NaCl, 0.1% Tween (freshly added)), 2x in EB (10 mM Tris-HCl pH 8.0), and 1x T4 DNA Ligase Buffer (NEB). Dynabeads were resuspended in end-repair reaction and incubated at 24C shaking at 800 rpm for 30 min. Dynabeads were washed 1x in 1X W&B, 2x EB, and 1x NEBuffer 2 (NEB) and resuspended in A-tailing reaction mix (NEB), followed by incubation at 37C shaking at 800 rpm for 30 min. Dynabeads were washed 1x in NEBuffer 2 and resuspended in ligation reaction mix (NEB), 5 nM annealed TruSeq truncated adapter and incubated at 25C shaking at 600 rpm for 20 min. Ligation reaction was stopped by adding 50 mM EDTA, and Dynabeads were washed 3x in 1X W&B, 3x EB. PCR amplified for 16 cycles with Kapa HiFI HotStart Ready Mix using TruSeq index adapters. PCR products were separated from DynaBeads and cleaned using AMPure Beads XP. library concentrations were calculated by KAPA Library Quantification Kit for Illumina Platforms (Kapa Biosystems) and sequenced on Illumina NextSeq 550 (75 bp single read). In the indicated experiments C/EBPα-induced macrophages were treated with the following compounds: Aphidicolin (APH; 4 μM) 4 hours before EdU incubation. PARPi (Olaparib; 5 μM) and ddC, ddA, ddG or ddT (40 µM) were added 20 hours prior to harvest. For i3Neurons, 40 µM of ddA, ddT, ddG or ddC chain-terminating nucleosides, 40 µM Ara-A and 20 µM Ara-C were added to cells for 18 hours prior to cell harvesting for SAR-seq experiments. 40 µM Gemcitabine was added to cells 2 hours prior to cell harvesting for SAR-seq experiments.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
EdU incorporated DNA
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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.
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
Assembly: hg19 for human, mm10 for mourse.
Library strategy: SAR-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 |
GPL21697 |
| Series (2) |
| GSE210312 |
Active DNA demethylation promotes cell fate specification and the DNA damage response [SAR-Seq] |
| GSE210317 |
Active DNA demethylation promotes cell fate specification and the DNA damage response |
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| Relations |
| BioSample |
SAMN30102034 |
| SRA |
SRX16769848 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6428025_iNeuron_ddA_SAR-seq.bw |
124.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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