 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 01, 2024 |
| Title |
Control-Replicate 2 |
| Sample type |
SRA |
| |
|
| Source name |
JM8.N4
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
cell line: JM8.N4
cell type: mESC
treatment: Control
|
| Treatment protocol |
The plant auxin analog Indole-3-acetic acid (IAA) sodium salt was dissolved in ddH2O with a stock concentration of 500mM, aliquoted and stored at −20°C. To completely deplete endogeneous RAD21, cells were treated with IAA at a concentration of 500µM for 6 hrs.
|
| Growth protocol |
JM8.N4 mouse embryonic stem cells (mESCs) from the C57BL/6N strain and their genome-edited derivatives were routinely cultured in 60mm plates coated with 0.1% gelatin without feeders at 37°C and 5% CO2. The mESC culture medium was composed of the optimized knockout DMEM for mESCs, 15% ESC-qualified Fetal Bovine Serum, 1000 units of Leukemia inhibitory factor (LIF) , 1mM GlutaMAX , 0.1mM MEM nonessential amino acids , 0.1mM β-mercaptoethanol and Antibiotic-Antimycotic . 2i inhibitors were also added into the medium at final concentrations of 1uM for PD0325901 and 3uM for CHIR99021, respectively.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
1ul harsh lysis buffer (50mM Tris (pH=8.0), 5mM EDTA (pH=8.0), 10mM DTT, 1% Tween-20, 1% Triton X-100, 0.1g/l proteinase K, 2.5mM dNTPs, and ERCC Mix (107-fold dilution) and 1ul 10mM barcoded RT primer was added to each well. Plates were incubated at 50°C for 5 mins to lyse cells and proteinase K was heat-inactivated by subsequently incubating at 80°C for 20 mins. To minimize contamination across wells, heavy-duty plate seals and qPCR compression pads (Thermo Fisher Scientific, Cat. 4312639) were used to seal the plates. The lysis reaction was mixed with 2ul reverse transcription master mix 5×buffer (Thermo Fisher Scientific, Cat. 11756500), 2ul 5M Betaine (Sigma-Aldrich, Cat. B0300-1VL), 0.2ul 50mM E5V6NEXT template switch oligo (Integrated DNA Technologies), 0.1ul 200U/ul Maxima H-RT (Thermo Fisher Scientific, Cat. EP0751), 0.1ul 40U/ul NxGen RNase Inhibitor, and 0.6ul nuclease-free water (Thermo Fisher Scientific, Cat. AM9932). The reaction system was then incubated at 42°C for 1.5 hrs, followed by 10 mins at 75°C to inactivate reverse transcriptase. PCR was performed by adding 10ul 2×HiFi PCR mix (Kapa Biosystems, Cat. 7958927001) and 0.5ul 60mM SINGV6 primer and running the following program: 98°C for 3 mins, 20 cycles of 98°C for 20 s, 64°C for 15 s, 72°C for 4 mins, with a final extension step off 5 mins at 72°C.
Mouse embryonic stem cells treated with or without auxin were washed and resuspended in 1xPBS with 0.04% BSA. Nuclei isolation for single-cell ATAC (scATAC) sequencing from cell suspensions was carried out according to the manufacturer’s demonstrated protocol (Demonstrated Protocol, CG000169, Rev E,10xGenomics). Nuclei were counted using a Luna-II automated cell counter (Logos Biosystems). Approximately 15,000 nuclei per sample were loaded and subjected to the Chromium NextGem scATAC-seq v2 assay (10X Genomics).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic single cell |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
| |
|
| Description |
10XGenomics
|
| Data processing |
Libraries were sequenced on a NextSeq 2000 (Illumina) with 50bp read 1, 8bp i7 index read, 16bp i5 index read, and 50bp read 2. FASTQ files of raw data were processed by Cell Ranger ATAC (10xGenomics, v2.1.0) analysis pipeline. Reads were filtered and aligned to mouse genome mm10 (10xGenomics, refdata-cellranger-arc-mm10-2020-A-2.0.0) using cellranger-atac count() function with default parameters. Then the barcoded and aligned fragment files were loaded by ArchR (version 1.0.1). Low quality cells with minimum TSS enrichment score less than 4 and minimum fragment number less than 1000 were filtered out. Doublets were inferred by addDoubletScores() function and removed using filterDoublets() function with default parameters.
Assembly: mm10
Supplementary files format and content: Tab-separated value and text files
|
| |
|
| Submission date |
Apr 29, 2024 |
| Last update date |
May 01, 2024 |
| Contact name |
PENG DONG |
| E-mail(s) |
p.dong@siat.ac.cn
|
| Organization name |
Shenzhen Institute of Advanced Technology
|
| Street address |
1068 Xueyuan Avenue, Shenzhen University Town
|
| City |
Shenzhen |
| State/province |
Guangdong |
| ZIP/Postal code |
518055 |
| Country |
China |
| |
|
| Platform ID |
GPL30172 |
| Series (1) |
| GSE266089 |
Single-cell ATAC-seq analysis of mouse embryonic stem cells before and after Cohesin depletion |
|
| Relations |
| BioSample |
SAMN41111067 |
| SRA |
SRX24390828 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8240336_Sample_10xATAC_mESC_control_rep2_barcodes.tsv.gz |
30.9 Kb |
(ftp)(http) |
TSV |
| GSM8240336_Sample_10xATAC_mESC_control_rep2_filtered_peak_bc_matrix.h5 |
94.5 Mb |
(ftp)(http) |
H5 |
| GSM8240336_Sample_10xATAC_mESC_control_rep2_matrix.mtx.gz |
179.5 Mb |
(ftp)(http) |
MTX |
| GSM8240336_Sample_10xATAC_mESC_control_rep2_peak_annotation.tsv.gz |
2.5 Mb |
(ftp)(http) |
TSV |
| GSM8240336_Sample_10xATAC_mESC_control_rep2_peaks.bed.gz |
1.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
