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| Status |
Public on Mar 03, 2024 |
| Title |
2i_ESCs_RNAseq_rep2 |
| Sample type |
SRA |
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| Source name |
E14 cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
culture method: 2i
genotype: wildtype
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| Growth protocol |
E14Tg2a (E14) ESCs were cultured in standard culture medium on gelatin-coated dishes without feeder cells: For serum/LIF culture, DMEM high-glucose media supplemented with 15%FBS, 1 mM sodium pyruvate (Gibco), 1% nonessential amino acids (Gibco), 1% GlutaMAX (Gibco), 0.1 mM β-mercaptoethanol (Life Technologies), 1% penicillin-streptomycin (Gibco)，1000 U/mL LIF (Millipore). For 2i/LIF culture, mouse ESCs were cultured in 2i medium as previously described2. In total, 500 mL of 2i medium were prepared with 240 mL of DMEM/F12 (Thermo Fisher Scientific), 240 mL of neurobasal (Thermo Fisher Scientific), 2.5 mL of N2 supplement (Thermo Fisher Scientific), 5 mL of B27 supplement (Thermo Fisher Scientific), 1% GlutaMAX, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol, and 1% penicillin-streptomycin, 1000 U/mL LIF, 3 μM CHIR99021 (Selleck), and 1 μM PD0325901 (Selleck). All cells were maintained at 37°C in an incubator with 5% CO2. The medium was changed every day, and the cells were passaged with 0.25% trypsin-EDTA (Gibco). Interconversion of 2i- and serum-ESCs: 2i- and serum-ESCs were cultured on gelatinized plates for 3 days, and were digested with 0.25% trypsin, then 2i-ESCs or serum-ESCs were plated into gelatinized six-well plates with 6-7 × 104 cells per well in serum/LIF or 2i medium. Then change the medium every day.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-seq, total RNAs were extracted with TRIzol® reagent. RNA sequencing libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit (Vazyme Biotech). The libraries were denatured and diluted at a proper concentration, then were sequenced on Illumina NovaSeq (Annoroad Gene Technology Co., Ltd). For ATAC-seq, 50,000 cells were harvested and washed once with 50 μL cold PBS. Then the cells were resuspended in 50 μL lysis buffers (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). Then the suspension of nuclei was centrifuged 500 × g at 4°C for 10 min. The pellet was resuspended with 50 μL transposition reaction mix (10 μL TD buffer, 5 μL Tn5 transposase and 35 μL nuclease-free H2O), and incubated at 37°C for 30 min. Finally, DNA was isolated using MinElute PCR Purification Kit (QIAGEN). ATAC-seq libraries were constructed and were purified with AMPure XP beads (Beckman Coulter). The libraries were denatured and diluted, then were sequenced on HiSeq X-Ten (Annoroad Gene Technology Co., Ltd). For ChIP-seq,1 × 107 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 × 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 antibody–Dynabeads protein A/G (1:1 mixed) at 4°C overnight with rotation. Antibodies included：anti-YY1 antibody (Abcam, ab109237)，anti-SMC1 antibody (Bethyl Laboratories, A300-055A).ChIP For Biotin ChIP, cells stably expressed biotin-alone or biotin-TEAD2 were expanded and crosslinked with 1% formaldehyde. Crosslinked cells were sonicated and diluted tenfold with ChIP dilution buffer, and then incubated with M280 streptavidin dynabeads at 4°C for overnight. Streptavidin dynabeads-bound DNA was subsequently washed twice with wash buffer 1 (2% SDS), once with wash buffer 2 (50 mM HEPES (pH 7.5), 1 mM EDTA, 500 mM NaCl, 0.1% sodium deoxycholate, 1% Triton X-100), once with wash buffer 3 (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate) and then twice with TE wash buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). ChIPed DNA was reverse-crosslinked and purified for DNA library construction followed by sequencing. For BL-HiC, the cells were treated with 1% formaldehyde at room temperature for 10 min followed by quenching with 0.2 M glycine. Nuclei were extracted for subsequent experiments, including digestion with HaeIII (NEB), end-plus-A treatment, proximity ligation with biotin-labeled Bridge Linker, DNA purification and enrichment of biotin-labeled DNA with Dynabeads M-280 streptavidin. The enriched bead-bound DNA was subjected to end repair, adapter ligation, PCR amplification and DNA library construction, followed by sequencing on the Illumina NovaSeq platform (Annoroad Gene Technology Co., Ltd.). For QHR-4C, digested suspensions of 1 × 105 - 1 × 106 cells from tissues were cross-linked with 2% formaldehyde for 10 min and terminated by 0.2 M glycine. The cell pellet was permeabilized and digested with DpnII overnight followed by proximity ligation. Then, DNA under 1000 bp was extracted and sonicated. To enrich ligation events associated with a specific viewpoint, an appropriate amount of sonicated DNA was taken as a template to linearly amplify for 100 cycles using a 5’ biotin-labeled probe of the viewpoint of interest. The amplification products were incubated at 95°C for 5 min, immediately cooled on ice to obtain amplified ssDNA and then enriched with Dynabeads M-280 streptavidin. The bead-bound ssDNA was then ligated with adapters. Finally, QHR-4C libraries were constructed with specific primer pairs (forward primers containing Illumina P5 with sequences near a specific viewpoint and reverse primers containing Illumina P7 with an index and sequences matching the adapter) and then sequenced on the Illumina NovaSeq platform (Annoroad Gene Technology Co., Ltd.).
RNA sequencing libraries were constructed using the VAHTS mRNA-seq V3 Library Prep Kit for Illumina (Vazyme). ATAC libraries were constructed using the VAHTS Universal DNA Library Prep Kit for Illumina V3 (Vazyme). BL-Hi-C libraries were constructed into standard illumina libraries. ChIP-seq libraries were constructed using the VAHTSTM Universal DNA Library Prep Kit for Illumina® V2. QHR-4C libraries were constructed into standard illumina libraries.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Data processing |
Illumina Casava software used for basecalling.
Reads were aligned to the mm10 genome using bowtie2 version 2.3.2.
For RNAseq data, the reads were qualified with FastQC tool and trimmed with trim_galore if reads contained adapters. Processed reads were mapped to Ensembl transcriptome version 95 (mm10) by using RSEM . Raw tag counts were extracted from sample.gene.results files and merged together, and then GC-normalized using EDASeq.
For ATACseq data, reads were aligned to mm10 genome by using Bowtie2 with default parameters. Reads mapping to mitochondrial DNA or unassigned sequences were discarded. Finally, concordantly aligned pairs were retained. The BAM files of biological replicates were merged, and peaks were called by using dfilter with the settings of -bs=100 -ks=50 -pe -lpval=2 . Alignment BAM files were normalized by RPKM and transformed into read coverage files (bigWig format) by using deepTools.
For ChIPseq data, reads were aligned to mm10 genome by using Bowtie2 with default parameters. Reads mapping to mitochondrial DNA or unassigned sequences were discarded. Finally, concordantly aligned pairs were retained. The peaks were called by using MACS2 with the default parameters. Alignment BAM files normalized by RPKM and transformed into read coverage files (bigWig format) by using deepTools.
For BL-Hi-C data, were processed with ChIA-PET2 v0.9.2 software with the parameter -A ACGCGATATCTTATC -B AGTCAGATAAGATAT -s 1 -m 1 -t 4 -k 2 -e 1 -l 15 -S 500 to identify chromatin interactions that annotated with genome mm10. The biological replicates in each group were merged to perform the A/B compartments and TAD analysis. The interaction matrix was generated by HiC-Pro. Normalization was performed by HiCExplorer hicCorrectMatrix (--correctionMethod KR). TADs and TAD boundaries were defined by HiCExplorer hicFindTADs (--correctForMultipleTesting fdr --thresholdComparisons 0.05 --minDepth 120000 --maxDepth 200000 --step 40000) at 40-kb resolution. Compartments were analyzed using HOMER (v.4.10) tools with default parameters at 100 kb resolution. APA analysis was performed using juicer tools.There producibility of BL-Hi-Cdatasets was calculated on pairs of raw Hi-C contact matrices at 100 kb resolution by using HiCRep.
For 4C data, raw paired-end reads were removed with Trim Galore and then subjected to cutadapt (version 3.4) to trim the primer sequence at the 5 end of read 1. Reads that did not contain primer sequences were discarded. Reads were mapped to the mm10 genome using bowtie2 with the following parameters: --very-sensitive --end-to-end --no-unal -X 2000. Bam files were imported into the r3Cseq package (version 1.38.0). Normalized bedgraph files were thus generated and then transformed into bigwig files using the bedGraphToBigWig tool.
Assembly: GRCm38.p6
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| Submission date |
Feb 28, 2023 |
| Last update date |
Mar 03, 2024 |
| Contact name |
xiaotao dong |
| E-mail(s) |
guo_rong@gibh.ac.cn
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| Organization name |
Chinese Academy of Sciences
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| Street address |
190 Kai Yuan Avenue
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| City |
guangzhou |
| ZIP/Postal code |
51530 |
| Country |
China |
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| Platform ID |
GPL21273 |
| Series (1) |
| GSE226316 |
TEAD2 mediates the ground-state pluripotency by chromatin looping |
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| Relations |
| BioSample |
SAMN33534575 |
| SRA |
SRX19530966 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7072919_2i_ESCs_RNAseq_rep2_count.txt.gz |
728.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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