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| Status |
Public on Mar 11, 2024 |
| Title |
Naive-H9_Cuttagseq_H3K9me3_rep2 |
| Sample type |
SRA |
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| Source name |
H9
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
cell type: ESC
genotype: WT
treatment: navie induced medium
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| Treatment protocol |
The medium was changed every other day. The dome-shaped colonies were dissociated with Accutase (Thermo Fisher Scientific, catalog. no. A1110501) into single cells, and then passaged onto new Matrigel- or feeder-coated plates every 3 - 4 days.
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| Growth protocol |
The naive hESCs (also named as prEpiSCs) were derived from the LTR7-GFP hESC_H9 cell line. The basal culture medium (500 ml) is prepared as follows, 240 ml DMEM/F12 (Thermo Fisher Scientific, catalog. no. 21331020), 240 ml Neurobasal medium (Thermo Fisher Scientific, catalog. no. 21103049), 2 mM L-glutamine (Thermo Fisher Scientific, catalog. no. 25030081), non-essential amino acids (Thermo Fisher Scientific, catalog. no. 11140050), 1% N2 supplement (Thermo Fisher Scientific, catalog. no. 17502048), 2% B27 supplement (Thermo Fisher Scientific, catalog. no. 12587010, minus vitamin A), 100 mg/ml Vitamin C (Sigma, catalog no. A4403), 50 ng/ml Bovine Albumin Fraction V (Thermo Scientific, cat. no. 15260-037), 20 ng/ml IL6 (PeproTech, catalog. no. AF-200-06), 20 ng/ml sIL-6R (PeproTech, catalog. no. 200-06R), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, catalog. no. 21985023), Primocin (InvivoGen, catalog. no. ant-pm-2). The naive/prEpiSCs were cultured in the advanced GIX medium (basal culture medium plus chemical cocktail [0.2 μM Trametinib (GSK112022, Selleck S2673), 0.3 μM PD0325901, 2.0 μM XAV939, 0.5 μM Go6983]).IL6, IL-6R, Trametinib, PD0325901, XAV939, Go6983
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
the sample used to perform RNA-seq, ATAC-seq, CUT&Tag-seq are prepared based on the commonly used method.
as for CUT&Tag, 20000~50000 cells per sample replicate were washed in Wash Buffer [1 mL 1 M HEPES pH 7.5 (Sigma–Aldrich, H3375), 1.5 mL 5 M NaCl (Sigma–Aldrich, S5886-1KG), 12.5 μL 2 M Spermidine (MCE, HY-B1776), Roche Complete Protease Inhibitor EDTA-Free tablet (Sigma–Aldrich, 4693132001), and bring the final volume to 50 mL with dH2O], then immobilized on 10 ul of Concanavalin A-coated beads (Bangs Laboratories, BP531). Cells were cleared on a magnetic rack, then permeabilized with cold Antibody buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 2mM EDTA, 0.1% BSA, 0.5 mM Spermidine and 1× protease inhibitor cocktail containing 0.05% Digitonin (AbMole, M5020-100mg)] on ice. The cells were then incubated with anti-H3K9me3(Abcam, ab8898, 1:100 dilution), anti-H3K9ac (Abcam, ab32129, 1:100 dilution), anti-H3K27me3(Millipore, 07-449, 1:100 dilution), H3K27ac (Abcam, ab177178, 1:100 dilution), anti-CTCF (Millipore, ab128873,1:100 dilution) for 2h at RT on a shaker. The primary antibody was cleared on a magnetic rack. Goat anti-Rabbit IgG secondary antibody (Millipore, 07-449) was diluted 1:100 in Dig-wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine and 1× protease inhibitor cocktail containing 0.05% Digitonin) and incubated at RT for an hour. Cells were cleared on a magnetic rack and washed three times with 700ul of Dig-wash buffer. A 1:100 diluted of pA-Tn5 adapter complex combining adapter primers (Table S3) and pA-Tn5 according to the manufacturer’s instructions (Vazyme, S603-01) was prepared in Dig-300 Buffer [20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine and 1× protease inhibitor cocktail containing 0.05% Digitonin]. Cells were cleared on a magnetic rack and incubated by adding 100 ul of pA-Tn5 at RT for 1 h. Cells were washed with 700ul of Dig-300 buffer, resuspended in 300 ul of Tagmentation buffer [10 mM MgCl2 in Dig-300 Buffer], and incubated at 37 °C for 1 h. 10 ul of 0.5 M EDTA, 3 ul of 10% SDS, and 2.5 ul of 20 mg/mL Proteinase K was added to each reaction to stop the tagmentation at 37 °C overnight. DNA was purified using phenol/chloroform/isoamyl alcohol (PCI) extraction followed by chloroform extraction and precipitated with glycogen and ethanol. DNA was pelleted with a high-speed spin at 4 °C, washed, air dried for 5 min and resuspended in 25 ul of double-distilled water (ddH2O) containing 100ug/ml RNase. The DNA was then PCR amplified using TruePrep Index Kit V4 for Illumina (Vazyme, TD204), and cleaned up with VAHTS DNA Clean Beads (Vazyme, N411-01).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
Use FastQC v0.11.9 for quality control of raw sequencing readings.
Using TrimGalore v0.6.6 to remove raw readings from low-quality base and linker sequences
Clean reads of were mapped to hg38 using Bowtie2(2.3.1) The options are: --local --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700
Use the sorting function of samtools v1.13 to sort aligned bam files based on chromosome coordinates
Use the genomecov function of bedtools v2.30 to summarize the sorted bam files into a bedgraph file (Quinlan et al., 2010).
Peak calling on all bedgraph files using SEACR v1.3 by selecting the top 1% of the call peak.
Assembly: hg38
Supplementary files format and content: cut&tag peak bigwig files
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| Submission date |
Mar 06, 2024 |
| Last update date |
Mar 11, 2024 |
| Contact name |
Niannian Li |
| Organization name |
Nankai University
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| Department |
college of Life Sciences
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| Street address |
No. 94 Weijin Road, Nankai District, Tianjin
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| City |
Tianjin |
| State/province |
Tianjin |
| ZIP/Postal code |
300071 |
| Country |
China |
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| Platform ID |
GPL18460 |
| Series (2) |
| GSE260993 |
Single-cell 3D genome structure reveals distinct human pluripotent states (CUT&Tag-seq) |
| GSE260995 |
Single-cell 3D genome structure reveals distinct human pluripotent states |
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| Relations |
| BioSample |
SAMN40286028 |
| SRA |
SRX23855188 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8129343_Naive_H3K9me3_20_FKDL210115755-1a-AK3126.bw |
56.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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