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Sample GSM5320185

Query DataSets for GSM5320185

Status

Public on Oct 26, 2021

Title

E13.5_EC_2Runx1_ATAC_rep2

Sample type

SRA

Source name

Endothelial Cells

Organism

Mus musculus

Characteristics

cell type: Endothelial Cells
isolation markers: CD41-CD45-Ter119-CD31+VEC+cKIT lo/+
strain: C57BL/6J-129Sv/J
developmental stage: E13.5
organ: whole fetus
genotype: +2Runx1 (Cre; CKI/CKI)

Treatment protocol

Females were paired with males for timed matings. Females were injected IP with 2mg tamoxifen 24 hours prior to harvesting. Embryos were harvested at the appropriate embryonic day and cells were sorted on the same day as harvest.

Growth protocol

N/A

Extracted molecule

genomic DNA

Extraction protocol

50,000 cells were FACS sorted then washed with cold PBS. Cell pellet were resuspended in 50 µl of cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% (v/v) Igepal CA-630) and immediately applied to centrifugation at 1,600g, 4 °C for 10 min. Nuclei pellet was resuspended in 50 µl of transposition reaction mix (1× tagment DNA Buffer, 2.5 µl of Tagment DNA Enzyme 1) and incubated for 30 min at 37 °C.
Libraries were purified using a Qiagen MinElute Gel Purification kit and the concentrations were measured using both Qubit and KAPA qPCR. 2100 Bioanalyzer was used to check the quality of libraries. Libraries were sequenced on the Illumina NextSeq 500, with 75-bp paired-end reads.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Description

E13_2Runx1_ATAC.bw
E13_2Runx1_ATAC_peaks.bed

Data processing

ATAC-seq: Sequencing reads were demultiplexed using Bcl2Fastq v2.20 then trimmed and filtered for quality using Trim Galore v0.6.4 with the following settings: fastqc, paired, and trim1. Reads were then aligned to the mouse genome (mm10) using bowtie 2.3.5.1. Only uniquely mapped reads with fewer than 2 mismatches were used for downstream analyses. Samtools v1.1 was used to convert SAM files to BAM files, and Sambamba v0.6.6 was used to filter out duplicates, multi-mappers, reads mapped to ChrM or blacklist regions, and unmapped reads. MACS2 2.1.4 was used for peak calling using the following parameters: BAMPE, q: 0.05. . Merged replicates were used to create bigwig files for visualization using Deeptools v3.3.0 and the following parameters: normalized to reads per genomic content (RPGC), effective genome size: 2308125349, ignore for normalization: ChrX, min fragment length: 20, bin size: 10.
RNA-seq: Sequencing reads were demultiplexed using Bcl2Fastq then trimmed and filtered for quality using Trim Galore v0.6.4 with the following settings: fastqc, paired, and trim1. Reads were then aligned to the mouse genome (mm10) using STAR 2.7.3a and the mm10 gene reference file from UCSC. Only uniquely mapped reads with fewer than 2 mismatches were used for downstream analyses. Samtools v1.1 was used to convert to BAM files, and Sambamba v0.6.6 was used to filter out duplicates, multi-mappers, and unmapped reads. The featureCounts function of the Subread v2.0.0 package was used to extract gene-level and transcript level read counts. EdgeR and TMM normalization were used to calculate normalized RPKM. Bigwig files for visualization were created using Deeptools v3.3.0 from merged BAM files. The following parameters were used: normalized to reads per genomic content (RPGC), effective genome size: 2308125349, ignore for normalization: ChrX, min fragment length: 20, bin size: 10.
ChIP-seq: Sequencing reads were demultiplexed using Bcl2Fastq v2.20 then trimmed and filtered for quality using Trim Galore v0.6.4 with the following settings: fastqc, paired, and trim1. Reads were then aligned to the mouse genome (mm10) using bowtie 2.3.5.1. Only uniquely mapped reads with fewer than 2 mismatches were used for downstream analyses. Samtools v1.1 was used to convert SAM files to BAM files, and Sambamba v0.6.6 was used to filter out duplicates, multi-mappers, reads mapped to ChrM or blacklist regions, and unmapped reads. MACS2 2.1.4 was used for peak calling using the following parameters: broad, q: 0.05. Merged replicates were used to create bigwig files for visualization using Deeptools v3.3.0 and the following parameters: normalized to reads per genomic content (RPGC), effective genome size: 2308125349, ignore for normalization: ChrX, min fragment length: 20, bin size: 10, smooth length: 60, extend reads: 150.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: BigWig files provided are normalized tracks of merged replicates.
Supplementary_files_format_and_content: Bed files provided are the output of MACS2 peak calling.
Supplementary_files_format_and_content: RPKM_Norm.txt: Tab-delimited text file for RNA-seq samples includes normalized RPKM.

Submission date

May 17, 2021

Last update date

Oct 26, 2021

Contact name

Kai Tan

E-mail(s)

[email protected]

Organization name

Children's Hospital of Philadelphia

Street address

3501 CIVIC CENTER BLVD