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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 31, 2023 |
| Title |
RNA-seq of Dek-KO E14TG2a cells complemented with HA-Dek-R58/V63/R653A repeat 2 |
| Sample type |
SRA |
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| Source name |
E14TG2a
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14TG2a
cell type: mouse embryonic stem cell
genotype: Dek-KO; HA-Dek-R58/V63/R653A over-expession
treatment: none
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| Growth protocol |
Mouse E14TG2a ESCs (a kind gift from M. Zhang’s laboratory, Bioland Laboratory, Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, China) were cultured on gelatin-coated dishes in DMEM (High Glucose, with Sodium Pyruvate, with L-Alanyl-L-Glutamine) (Vivacell) supplemented with 15% fetal bovine serum (Vistech), 1× non-essential amino acids (Gibco), 100 μM β-mercaptoethanol (Sigma), and 1000 U/mL of leukemia inhibitory factor (LIF) (Millipore). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The CUT&Tag was performed following the protocol provided by the Hieff NGS® G-Type In-Situ DNA Binding Profiling Library Prep Kit (Yeasen). A total of 50,000 cells were harvested and subjected to two rounds of washing using washing buffer (composed of 20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, and 1× Protease inhibitor cocktail). Subsequently, the cells were incubated with activated Concanavalin A coated magnetic beads. To this, 0.5 μg of HA antibody was added and allowed to incubate on a rotating platform at 4 °C for a duration of 16 hours. Following this, 0.5 μL of Goat anti-Rabbit secondary antibody was introduced and incubated for an additional hour. After a thorough washing step, the cells were treated with pA/G-Tn5 transposase at 37°C for 1 hour. Subsequently, DNA extraction was carried out by DNA selection beads
Total RNA was harvested using Trizol reagent from 5*10^6 ESCs.
To amplify libraries, 16 μL CUT&Tag DNA was mixed with 1 μL of a uniquely barcoded i5, 1 μL a uniquely barcoded i7 primer, using a different barcode for each sample, 7 μL ddH2O, and 25 μL 2× Ultima Amplification Mix, and run the following cycling conditions with lid heat on: 72 °C for 3 min; 95 °C for 30 s; 12 cycles of 95 °C for 10 s, 55 °C for 30 s and 72 °C for 30 s; final extension at 72 °C for 5 min and hold at 4 °C. Post-PCR clean-up was performed by adding 1.2× volume of DNA selection beads (60 μL), and libraries were incubated with beads for 5 min at RT, washed twice gently in 80% ethanol, and eluted in 20 μL EB buffer.
RNA libraries were prepared for sequencing using standard
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
HBM_mut_RNAseq_rep2
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| Data processing |
For CUT&Tag analysis, the CUT&Tag Fastq read files were aligned to the mm10 reference genome using Bowtie2, utilizing the default parameters. To identify Dek-wt peaks, biological replicates were merged, and peak calling was carried out using MACS2 (version 2.2.6) with specific settings (--keep-dup all -g mm -P-value ≤ 1e–5) against the IgG data. The generation of CUT&Tag bedgraph files involved the subtraction of IgG signals from the experimental group signals using Deeptools' bamCompare, with the inclusion of --skipNAs and --scaleFactorsMethod readCount options. The CUT&Tag bedgraph files were then subjected to normalization by calculating the reads per million (RPM), and the resulting bigwig files were generated using bedGraphToBigWig.
For RNA-seq analysis, the RNA-seq data underwent initial quality assessment and adapter trimming utilizing Trim Galore (version 0.6.4_dev). Subsequently, the trimmed reads were aligned to the mm10 reference genome using STAR (version 2.7.2d). For the quantification of reads corresponding to genomic features, featureCounts (version 2.0.0) was employed.
Assembly: mm10
Supplementary files format and content: bigWig files were generated using bamCoverage.
Supplementary files format and content: narrowPeak files were generated using MACS v2
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| Submission date |
Nov 15, 2023 |
| Last update date |
Dec 31, 2023 |
| Contact name |
shen yunfan |
| E-mail(s) |
shenyunfan.csu@outlook.com
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| Organization name |
central south university
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| Street address |
xiangya road
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| City |
changsha |
| State/province |
hunan |
| ZIP/Postal code |
410000 |
| Country |
China |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE247796 |
Structural basis of the DEK-nucleosome complex and its functional implications |
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| Relations |
| BioSample |
SAMN38262199 |
| SRA |
SRX22530746 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7900902_HBM_mut_2.coding_genes.count.simple.txt.gz |
187.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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