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| Status |
Public on Sep 06, 2023 |
| Title |
Nanog-ChIP-seq-KOF6-Rep2-SL |
| Sample type |
SRA |
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| Source name |
embryonic
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| Organism |
Mus musculus |
| Characteristics |
tissue: embryonic
cell line: E14Tg2a
cell type: Embryonic stem cells
genotype: SPIC-KO+FLAG-NANOG
treatment: SL
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| Growth protocol |
mESCs (E14Tg2a) were routinely cultured in serum+LIF (SL) condition, on 0.1% gelatine-coated plates. To make SL medium, High glucose DMEM (Gibco) was supplemented with 14% heat-inactivated FBS (Gibco), 1% Glutamax (Gibco), 1% Na-pyruvate (Gibco), 1% penicillin/streptomycin (Gibco), 55 µM Bmercaptoetanol, and LIF (1,000 U ml-1, Millipore). For serum-free 2iL condition, cells were cultured on gelatine-coated dishes in NDiff 227 (TaKaRa) media supplemented with penicillin/streptomycin, LIF, and two inhibitors, GSK3 inhibitor CHIR99021 (Axon, 3 μM) and MEK inhibitor PD0325901 (Axon, 1 μM). For single inhibitors treatment experiments, only the noted inhibitor was omitted from the complete 2iL medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
mESCs were cultured in SL or 2iL medium for 3 days. For each replicate, 15 million cells were fixed with 1% paraformaldehyde. Fixed cells were consecutively treated with buffers A (148mM NaCl, 1.48 mM EDTA, 0.74 mM EGTA, 74mM HEPES), Buffer-B (10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, 0.25% Triton-x100), and Buffer-C (150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 50 mM HEPES). Isolated nuclei were lysed in freshly made sonication buffer (150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES, 0.15% SDS, 1% TritonX-100, 1X protease inhibitor) and samples were sonicated using Biorapture Pico (Diagenode). Sonicated chromatin was precleared using BSA blocked protein A/G beads (Invitrogen), and was further incubated with 10 µg of primary antibody (anti-FLAG from Sigma for NANOG ChIP, and anti-GFP from Abcam for SPIC-GFP ChIP). After overnight incubation, the immune complexes were captured by incubation with BSA blocked protein A/G beads for 2h on a rotating wheel at 4ºC. Beads were washed with ChIP wash buffer 1 (2 mM EDTA, 20 mM Tris pH:8 150 mM NaCl, 1% TritonX-100, 0.1% SDS), Buffer-2 (2 mM EDTA, 20 mM Tris pH:8 150 mM NaCl, 1% TritonX-100, 0.1% SDS, 500 mM NaCl) and Buffer-3 (1 mM EDTA, 10 mM Tris pH:8) for 10 minutes. Samples were then incubated with fresh ChIP elution buffer (0.1 M NaHCO3, 1% SDS) for 30 min at RT. Eluted chromatin was de-crosslinked overnight by adding 5M NaCl and proteinase K (final 10mg/ml) and incubating at 65ºC. DNA was purified using the MinElute PCR Purification Kit (Qiagen)
ChIP-Rx-seq libraries were subjected to library preparation using the KAPA HyperPrep kit (Roche) according to manufacturer’s instructions with dual indices and sequencing on an Illumina HiSeq 2500 sequencer (dual-indexed 50-bp paired-end reads).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
SPIC-KO+FLAG-NANOG-Rep2
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| Data processing |
FASTQ reads were mapped against mouse mm9 reference genome using Bowtie2. Reads were filtered for high quality mapping with SAMtools q>15 and PCR duplicates were removed using PicardTools. SAM files were converted to BAM using SAMTools. BAM files were converted to BED files using BEDTools, and reads were extended by 160 nt and converted to BedGraph using bamCoverage tool from the deepTools suite. BedGraphs were converted to BigWig files using bedGraphToBigWig from UCSC tool suite for further visualization using Integrative Genomics Viewer browser. Peak calling was performed using MACS2 58 and assigned peaks were filtered against the ENCODE black list regions. For read counting in assigned peaks, BAM files were first converted to BED files and read counting was performed using BEDTools. Read count across different samples were normalized for library size using DESeq2 (bioconductor.org) and values were used for differential peak analysis with the following criteria: minimum read number >20 reads per peak in at least one sample, P-Val<0.05, FC>=2). To identify genes in the vicinity to ChIP-seq/ ATAC-seq peaks, BEDTools window with -w 100,000 was used.
Assembly: mm9
Supplementary files format and content: xls, normalized ChIP-seq counts and peaks
Supplementary files format and content: bigwig
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| Submission date |
Jan 03, 2023 |
| Last update date |
Sep 06, 2023 |
| Contact name |
Yaser Atlasi |
| E-mail(s) |
y.atlasi@qub.ac.uk
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| Organization name |
Queen's University Belfast
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| Department |
patrick G Johnston Centre for Cancer research
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| Street address |
Lisburn Road
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| City |
Belfast |
| ZIP/Postal code |
BT9 7AE |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE222057 |
(ATAC-seq_and_ChIP-seq) Spic regulates one-carbon metabolism and histone methylation in ground-state pluripotency |
| GSE222058 |
Spic regulates one-carbon metabolism and histone methylation in ground-state pluripotency |
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| Relations |
| BioSample |
SAMN32541659 |
| SRA |
SRX18912268 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6914000_PN0442_S6.dn.bam.bg.bw |
114.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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