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Sample GSM7900916

Query DataSets for GSM7900916

Status

Public on Dec 31, 2023

Title

HA-CUT&Tag of Dek-KO E14TG2a cells complemented with HA-Dek-R58/V63/R653A+LRM_deletion repeat 2

Sample type

SRA

Source name

E14TG2a

Organism

Mus musculus

Characteristics

cell line: E14TG2a
cell type: mouse embryonic stem cell
genotype: Dek-KO; HA-Dek-R58/V63/R653A+LRM_deletion over-expession
treatment: HA CUT&Tag antibody

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.

Extracted molecule

genomic DNA

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

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

HBM_mut_LRM_Del_HA-Dek_CUT&Tag_rep2

Data processing

*library strategy: CUT&Tag
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

Submission date

Nov 15, 2023

Last update date

Dec 31, 2023

Contact name

shen yunfan

E-mail(s)

shenyunfan.csu@outlook.com

Organization name

central south university

Street address

xiangya road