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Sample GSM4229170

Query DataSets for GSM4229170

Status

Public on Dec 31, 2020

Title

DNase-seq_EILP_rep1_GB3053 (duplicate GSM3677394)

Sample type

SRA

Source name

EILP

Organism

Mus musculus

Characteristics

strain: C57BL/6
genotype/variation: Tcf7EGFPnull/+
tissue: bone marrow
sorting profile: Lin- Kit+ 2B4+ a4b7+ Tcf7+ Thy1-

Growth protocol

Samples were isolated by cell sorting from bone marrow single cell suspensions

Extracted molecule

genomic DNA

Extraction protocol

For DNase I digestion, 300 cells from each cell type were collected by FACS sorting. 0.3 unit of DNase I (Roche, 04716728001) was added to each cell type and incubated at 37 °C for 5 minutes. Reaction were stop by adding 80 μl of stop buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.15% SDS, 10 mM EDTA) containing 1 μl of 20 mg/ml proteinase K. Samples were incubated at 55 °C for overnight and DNA purified by phenol–chloroform extraction, followed by precipitation with ethanol in the presence of 20 μg glycogen.
The library was prepared using Illumina kits as described G. Hu et al. (Nature immunology 2013). The libraries were amplified using a two-step method to preferentially amplify the small DNA fragments derived from the DNA hypersensitive sites and to reduce non-specific amplification of the carrier DNA. The first amplification was done with index primers with the PCR condition: 98°C, 10"; 67°C, 30"; 72°C, 30" for 6 cycles. The second amplification was done with the P5 and P7 primers with the condition: 98°C, 10"; 68°C, 30"; 72°C, 30" for 22 cycles. The fragments between 160bp to 300bp were isolated on E-gel and sequenced on Illumina HiSeq3000.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 3000

Description

EILP_WT_DHS.se-W100-normalized.wig.gz

Data processing

Library strategy: DNAase-seq
Basecalling by RTA 1.18.66.3, Illumina instrument run time analysis software
Sequencing reads were aligned to mouse genome (mm9) using Bowtie2 with default parameters.
Reads from replicates were merged and redundant reads were removed from further analysis.
For ChIC-seq samples, reads from replicates were merged for peak calling. Peak calling were performed using MACS2 with default parameters.
For scMNase-seq samples, reads with length between 140 and 180 bp were selected as nucleosome reads while reads with length less than 80 bp were selected as subnucleosomal reads.
Genome_build: mm9
Supplementary_files_format_and_content: For ChIC-seq and DNase-seq samples, .wig files contain normalized tag density, which can be uploaded to UCSC genome browser for visulization. For scMNase-seq samples, bed files provide genomic positions on chromosomes for nucleosomes (140-180 bp) and subnucleosomes (<80 bp).

Submission date

Dec 20, 2019

Last update date

Dec 31, 2020

Contact name

Keji Zhao

Organization name

NHLBI,NIH

Department

Systems Biology Center

Lab

Laboratory of Epigenome Biology