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| Status |
Public on Apr 05, 2022 |
| Title |
D10_oeCTCF_shSmarca5_CTCF [ChIP-seq] |
| Sample type |
SRA |
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| Source name |
Reprogramming cells, OSKM plus CTCF, in addition to Smarca5 knockdown
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| Organism |
Mus musculus |
| Characteristics |
cell type: Reprogramming cells
day of reprogramming: D10
chip antibody: CTCF(Active motif Cat# 61311)
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| Treatment protocol |
20,000 MEFs at passage 2 were plated in a 12-well plate and then infected twice with retroviral stocks generated with Plat-E cells. 3 volumes of the supernatants of OSKM transcription factors were mixed with 1 volume of fresh MEF medium containing polybrene at a final concentration of 8 μg/ml. The infection efficiency was tested by MEFs transduced with virus of pMXs-EGFP. pMXs-CTCF and shRNAs were also transfected into OG2-MEFs by retrovirus generated from Plat-E cells. For infecting OG2-MEFs, the medium of OG2-MEFs was replaced with 2 ml of infection mixture per well in a 12-well plate. Infected cells were cultured with mESC medium containing 50 μg/ml Vitamin C (Sigma) post infection and renewed daily. For polycistronic OSKM-mediated reprogramming experiments, MEFs were transduced with 1 volume inducible lentivirus of OSKM and 2 volume rtTA retroviruses generated from HEK293T cells. Infected cells were cultured in mESC medium containing 50 μg/ml Vitamin C and 2 μg/ml doxycycline (Sigma) for 12 days to count GFP positive colonies. To ensure proper CTCF knockdown efficiency before transfecting these virus supernatants into MEFs, we concentrated virus supernatant produced from Ctcf shRNAs.
Lentivirus of lenti-birAV5 and pSIN-FLBio-SMARCA5 were assembled with psPAX2, pMD2.G vectors. mESCs were then infected with lenti-birAV5 lentivirus and selected with 10 μg/ ml of blastcidin for 5 days. BirAV5 overexpression was detected with anti-V5 antibody by Western blot. Then BirAV5-overexpressed cells were infected with pSIN-FLBio-Smarca5 lentivirus. Thereafter, cells were selected with 2 μg /ml of puromycin for 5 days, then single clones were picked up. Finally, in vivo biotinylation of SMARCA5 was detected with Anti-BIOTIN antibody (Cell Signaling Technology, CatLog: #7075) with a dilution ratio of 1:1000.
Three sets of oligonucleotides were cloned into an U6-sgRNA vector, separately, to create paired sgRNA target sites for SpCas9-nickase in the first or second exon of Smarca5. CRISPR/Cas9 target sites in Smarca5 (NM_053124.2) were designed by http://zifit.partners.org/ZiFiT/CSquare9Nuclease.aspx. The sequences of these three oligos were listed in Key Resources Table. Then these three sets of oligonucleotides were synthesized and inserted into the Bbs I site of the U6-gRNA vector. To generate stable Smarca5-/- mESC lines, two of the three gRNAs were used together, 2×10e6 mESCs were transfected with 20 μg of U6-sgRNA vector and 50 μg of pcDNA3.3-hCas9 plasmids. Cells were selected with 500 μg/ml of G418 (Sigma) after 24 hr of infection and were maintained in selection for 72 hr. Single clones were separated and were seeded in each well of a 48-well plate. Genotype of each colony was examined through Western blot.
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| Growth protocol |
mESCs and iPSCs were cultured in DMEM/High glucose medium (Hyclone) supplemented with beta-mercaptoethanol (10 mM), sodium pyruvate (10 mM), non-essential amino acids (10 mM), GlutaMAX (10 mM), 15% fetal bovine serum (Gibco), 1000 U/ml leukemia inhibitory factor (LIF), 1 μM MEK inhibitor PD0325901 and 3 μM GSK3 inhibitor CH99021. MEF cells were cultured in 10% FBS medium supplemented with non-essential amino acids (10 mM) and GlutaMAX (10 mM).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP experiments were performed as previously described. Briefly, 1×10e7 cells were crosslinked with 1% formaldehyde at room temperature for 10 min. Then the reaction was stopped by adding glycine (final concentration, 0.125 M). Crosslinked cells were lysed in ChIP SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl (pH 8.0)) containing 1 X Protease Inhibitor Cocktail and PMSF, then sonicated to achieve a chromatin sized of 200-400 bp. After sonication, the supernatant was diluted with IP buffer and then co-incubated with antibodies–Dynabeads protein A/G (1:1 mixed) at 4°C overnight with rotation. Immune complexes were washed with the following buffers: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.0), 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid (sodium salt), 1 mM EDTA, 10 mM Tris-HCl (pH 8.0)) and TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). Antibody-bound chromatin was reverse-crosslinked, and the ChIPed DNA samples were purified for ChIP-Seq library generation. The ChIPmentation experiments were performed as previously described. Briefly, 1×10e5 cells were crosslinked with 1% formaldehyde at room temperature for 10 min. Then the reaction was stopped by adding glycine (final concentration, 0.125 M). Crosslinked cells were lysed in lysis buffer (10 mM Tris-HCl,100 mM NaCl (pH 8.0),1 mM EDTA, 0.5 mM EGTA,0.1% NaDeoxycholate, 0.5% N-lauroylsarcosine, 1 x protease inhibitors), then sonicated to achieve a chromatin sized of 200-700 bp.Triton X-100 was added to a final concentration of 1%. After centrifuge, the supernatant was co-incubated with antibodies–Dynabeads protein A/G (1:1 mixed) at 4°C overnight with rotation. Immune complexes were washed with the following buffers: low salt wash buffer, high salt wash buffer, LiCl wash buffer, TE buffer and 10 mM Tris-HCl (pH 8.0). For tagmentation, bead bound chromatin was resuspended in 45 μl of tagmentation buffer, 5 μl of transposase (Vazyme) was added and samples were incubated at 37°C for 10 min followed by two washes with low-salt buffer. Antibody-bound chromatin was reverse-crosslinked, and the ChIPed DNA samples were purified for ChIP-seq library generation.
ChIP-seq sequencing libraries were constructed using the VAHTSTM Universal DNA Library Prep Kit for Illumina® V2.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
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| Data processing |
ChIP-seq raw reads were first assessed with FastQC tool and trimmed with trim galore if they contained adaptors.
Processed reads were mapped to the mm10 assembly using Bowtie2 with parameters “—very-sensitive –end-to-end –no-unal”. Any reads overlapped with mm10 blacklist regions (from ENCODE project) were filtered with bedtools, then proper-aligned and high-quality reads (MAPQ>30) were extracted with samtools, duplicated reads were removed using picard tools. Peaks were called by MACS2. Peak annotation, motif discovery, and the gene ontology analysis were performed using HOMER annotatePeaks.pl, or findMotifsGenome.pl. Differential binding sites were identified by MACS2bdgdiff tool with default parameters, or with glbase.redefine_peaks. Heatmaps from ChIP-seq was created with glbase or deeptools.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Peak files were generated using MACS2, coupled to redefine_peaks() from glbase3. Bigwig files were generated from normalized bdg files using bedGraphToBigWig tool.
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| Submission date |
Dec 11, 2018 |
| Last update date |
Apr 05, 2022 |
| Contact name |
ya wei song |
| E-mail(s) |
song_yawei@gibh.ac.cn
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| Phone |
13288824450
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| Organization name |
Guangzhou Institute of Biomedicine and Health,Chinese Academy of Sciences
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| Department |
South China Institute for Stem Cell Biology and Regenerative Medicine
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| Street address |
190 Kai Yuan Avenue, Science Park
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| City |
Guangzhou |
| State/province |
Guangdong |
| ZIP/Postal code |
510530 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE123651 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming [ChIP-seq] |
| GSE123670 |
CTCF Functions as an Insulator for Somatic Genes and a Structural Regulator for Pluripotent Genes during Reprogramming |
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| Relations |
| BioSample |
SAMN10581220 |
| SRA |
SRX5125727 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3508691_Mm_ctcf_D10_oeCtcf_shSmarca5.bed.gz |
591.9 Kb |
(ftp)(http) |
BED |
| GSM3508691_Mm_ctcf_D10_oeCtcf_shSmarca5.rp1.trim_treat_pileup.bw |
208.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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