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Sample GSM8112460

Query DataSets for GSM8112460

Status

Public on Mar 06, 2024

Title

H4K16ac-con 2

Sample type

SRA

Source name

oocyte

Organism

Mus musculus

Characteristics

antibody: H4K16ac
cell line: oocyte

Extracted molecule

genomic DNA

Extraction protocol

Oocytes were incubated in 50 µL antibody buffer; 0.5 μg antibody was added and the mixture was incubated at 4℃ overnight. After washing twice with dig-wash buffer, 50 µL dig-wash buffer with 0.2 µg secondary antibody was added and the mixture was incubated at room temperature for 1 h. After washing twice with 100 µL dig-wash buffer, 1 µL pG/A-Tnp and 49 µL dig-300 buffer were added, and the samples were incubated at room temperature for 1 h. Next, the samples were washed twice with 100 µL dig-wash buffer. We subsequently added 200 µL tagmentation buffer and incubated the samples at 37°C for 1 h.The reaction was stopped with 5 μl Proteinase K、100 μl Buffer L/B, and extraction with DNA Extract beads.
CUT&Tag was performed using the Hyperactive Universal CUT & Tag Assay Kit for Illumina (TD903, Vazyme).Oocytes were incubated in 50 µL antibody buffer; 0.5 μg antibody was added and the mixture was incubated at 4℃ overnight.After washing twice with dig-wash buffer, 50 µL dig-wash buffer with 0.2 µg secondary antibody was added and the mixture was incubated at room temperature for 1 h. After washing twice with 100 µL dig-wash buffer, 2 µL pA/G-Tnp and 49 µL dig-300 buffer were added, and the samples were incubated at room temperature for 1 h. Next, the samples were washed twice with 100 µL dig-wash buffer.We subsequently added 200 µL tagmentation buffer and incubated the samples at 37°C for 1 h. The reaction was stopped with 5 μl Proteinase K、100 μl Buffer L/B; After extraction with DNA Extract beads PCR was performed to amplify the libraries under the following conditions: 72°C for 3 min, 98°C for 30 s, 17 cycles of 98°C for 10 s and 60°C for 5 s, and a final extension at 72°C for 1 min with a hold at 4°C.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina NovaSeq 6000

Description

H4K16ac-con.bw

Data processing

CUT&Tag paired-end reads were aligned to the mouse genome (mm10) using Bowtie 2 (version 2.2.5) with a default setting after removing adaptor sequences and low-quality reads by fastp (version 0.12.4). Reads from PCR duplicates were removed using Picard ‘MarkDuplicates’. After confirming reproducibility between replicates, they were merged using Samtools‘merge’. Final bam files were converted to bigWig files of read coverages normalized to RPKM using deepTools (version 3.5.1) bamCoverage.
For RNA-seq analysis of all data, we downloaded the mouse reference genome (genome assembly: mm10) from the Ensembl database and used the HISAT2 software for read alignment with a default setting after removing adaptor sequences and low-quality reads by fastp (version 0.12.4). The gene-level quantification approach was used to aggregate raw counts of mapped reads using the featureCounts tool. The expression level of each gene was quantified in terms of the normalized fragments per kilobase of transcript per million mapped reads (FPKM). Next, we used the R package DESeq2 for differential gene expression analysis.
Assembly: mm10
Library strategy: Cut&Tag

Submission date

Feb 26, 2024

Last update date

Mar 06, 2024

Contact name

Ming Zong

E-mail(s)

ZongMing1112@126.com

Organization name

Wenzhou Medical University

Street address

University Town, Chashan, Wenzhou