GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM6428085 Query DataSets for GSM6428085
Status Public on Dec 01, 2022
Title iNeuron_POLBKD_ddC_S1ENDseq
Sample type SRA
Source name iNeuron
Organism Homo sapiens
Characteristics genotype: POLB knockdown
treatment: ddC, 18 hours prior to cell harvesting
dose: 40 µM
in-situ treatment: 1.8 U of S1 nuclease @15 mins on ice, followed by 37°C for 20 mins.
harvested day: day7
Treatment protocol In the indicated experiments cells were treated with the following compounds: For iMacrophages, Aphidicolin (APH; 4 μM) 4 hours before EdU incubation; PARPi (Olaparib; 5 μM) and ddC (20 µM) were added 20 hours prior to harvest. For chain-terminating nucleosides, 20 µM of each of ddA, ddT, ddG and ddC was added to cells for 18 hours prior to cell harvesting for END-seq and S1-END-seq.
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 pre-B-to-macrophage transdifferentiation, 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 For END-seq, live cells were harvested and embedded into agarose plugs (Bio-Rad cat#1703591). Cells were lysed in agarose using proteinase K, follwed by Rnase treatment. DNA was later precipitated after plug melting and shearing. For S1-END-seq, cells were collected and embedded in 1% agarose plugs, lysed and digested with Proteinase K (1 hour at 50°C, followed by 7 hours at 37°C), washed with TE buffer, and then treated with RNAse A for 1 hour at 37°C. Plugs were then washed in EB and equilibrated in S1 nuclease buffer (40 mM sodium acetate pH 5.2, 300 mM NaCl, 2 mM ZnSO4) for 30 minutes. 1.8 U of S1 nuclease was added to 100 µL of S1 nuclease buffer per plug and incubated on ice for 15 minutes to allow for the enzyme to diffuse into the plug. The reaction mix was then placed at 37°C for 20 minutes before addition of EDTA (10 mM final concentration) to terminate the reaction. Finally, plugs were processed through the standard END-seq protocol
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).
Library strategy OTHER
Library source genomic
Library selection other
Instrument model NextSeq 550
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.
bigwig files were made for positive (* and negativ (* strand separately using Bedtools genomecov function followed by UCSC toolkit function bedGraphToBigWig
Assembly: hg19 for human, mm10 for mourse.
Library strategy: END-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 GPL21697
Series (2)
GSE210314 Active DNA demethylation promotes cell fate specification and the DNA damage response [END-Seq]
GSE210317 Active DNA demethylation promotes cell fate specification and the DNA damage response
BioSample SAMN30102331
SRA SRX16769903

Supplementary file Size Download File type/resource 83.9 Mb (ftp)(http) BW 87.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap