 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 11, 2024 |
| Title |
Naive-H9_ATACseq_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
H9
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
cell type: ESC
genotype: WT
treatment: navie induced medium
|
| 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.
|
| 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
|
| 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 ATAC-seq, Cells were harvested and counted. Number of 500~1000 cells were used for ATAC-seq. The number of cells at this step is crucial, as the transposase-to-cell ratio determines the distribution of DNA fragments generated. Sample with 500~ 1000 cells was centrifuged for 5 min at 500 × g at 4 °C. Supernatant was removed and discarded. Cells were washed once with 50 μl of cold PBS buffer, centrifuged for 5 min at 500 × at 4 °C, and supernatant removed and discarded. The cell pellet in 50 μl of cold lysis buffer was resuspended by gently pipetting up and down and lysed for 10 min on ice, followed by centrifugation immediately for 10 min at 500 × g at 4 °C. The supernatant was discarded, and immediately continued to transposition reaction. Lysis buffer used was, 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40.For transposition reaction and purification, TruePrep® DNA Libraryaf Prep Kit V2 for Illumina (Vazyme, TD502, 5ng) for tagmentation was used. After tagmentation reaction, AMP XP beads for purifying was used. In preparation, AMPure XP beads were equilibrated at room temperature for 30 minutes and mixed well. 25 μl (0.6x) beads were pipetted into the 50 μl PCR product, gently pipetted and mixed, and incubated at room temperature for 5 min (to remove long fragments). The reaction tube was centrifuged briefly and placed on a magnetic stand. After clarification, the supernatant (75 μl) was transferred to a new tube (be careful to mark the tube cap and tube body), and the magnetic beads discarded. 60 μl (1.2x) beads were pipetted into the supernatant, mixed by pipetting, and incubated at room temperature for 5 min (magnetic beads can be added to the new EP tube during the first incubation). The reaction tube was centrifuged briefly and placed on a magnetic stand. After clarification, the supernatant was removed, the tube was kept on the magnetic stand. 200 μl of freshly prepared 80% ethanol was added to rinse the magnetic beads, incubated at room temperature for 30 s, and the supernatant carefully removed. The rinse was repeated three times. The tube was kept on the magnetic stand, the lid left open and dry (not too dry, no alcohol residue at the bottom of the tube). The tube was taken out of the magnetic stand, 25 μl of sterile ultrapure water added, the magnetic beads smashed from the wall, mixed well, and incubated at room temperature for 5 min. The reaction tube was dentrifuged briefly and placed on a magnetic stand. After clarification, 20 μl of supernatant was carefully transferred to a new tube and stored at −20°C.Library was constructed as follow. TruePrep TM Index V4 for Illumina (Vazyme TD204) was used to enrich DNA and PCR amplification. The PCR products were purified with VAHTS DNA Clean Beads (Vazyme N411). At last, DNA fragments of preferentially about 300 bp in length were selected, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA)
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 1500 |
| |
|
| Data processing |
ChiLin pipeline 2.0.0 (Qin et al., 2016) is used for QC and preprocess of the ATAC-seq.
We use Burrows-Wheeler Aligner (BWA) as a read mapping tool, and Model-based Analysis of ChIP-Seq (MACS2) as a peak caller, with a q-value (FDR) threshold of 0.01
Deeptools is used for the heatmap plots
Differential peaks between Naive and Primed were identified by DEseq2 with adjusted P ≤0.05, |log2fold change| ≥ 1.
Assembly: hg38
Supplementary files format and content: ATACseq peak bigwig files
|
| |
|
| Submission date |
Mar 06, 2024 |
| Last update date |
Mar 11, 2024 |
| Contact name |
Niannian Li |
| Organization name |
Nankai University
|
| Department |
college of Life Sciences
|
| Street address |
No. 94 Weijin Road, Nankai District, Tianjin
|
| City |
Tianjin |
| State/province |
Tianjin |
| ZIP/Postal code |
300071 |
| Country |
China |
| |
|
| Platform ID |
GPL18460 |
| Series (2) |
| GSE260991 |
Single-cell 3D genome structure reveals distinct human pluripotent states (ATAC-Seq) |
| GSE260995 |
Single-cell 3D genome structure reveals distinct human pluripotent states |
|
| Relations |
| BioSample |
SAMN40286120 |
| SRA |
SRX23855242 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8129287_ATAC-Naive-FKDL202618793.sorted.bam.bw |
25.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
