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Sample GSM1599176

Query DataSets for GSM1599176

Status

Public on Jul 21, 2015

Title

FL_LSK_Input

Sample type

SRA

Source name

Mouse Embryonic day 14.5 Fetal Liver

Organism

Mus musculus

Characteristics

strain: B6129SF1 crossed with C57BL/6
cell type: Fetal Liver, Lineage-Sca1+c-Kit+ (LSK)
chip antibody: None (input)

Treatment protocol

None

Extracted molecule

genomic DNA

Extraction protocol

[Isolation protocol] FLs were dissected from E14.5 embryos. Single cell suspensions were prepared by dissociating mechanically and expelling the cells through 40 μm Nylon filter, followed by red blood cell lysis (ACK Lysing Buffer, Lonza). To remove non-specific binding, anti-mouse CD16/CD32 (Fc Block, Biolegend) were added into single cell suspensions and incubated 10 min at 4°C. Next, Cells were stained with a cocktail of antibodies against several surface markers, including lineage markers (Ly-6G/Ly-6C, CD45R/B220, CD3ε, TER-119, CD4, CD8a, and CD19), CD117 (c-Kit), and Ly-6A/E (Sca-1). Cells were first subjected to yield sort for Lineage-Sca-1+c-Kit+ (LSK) and collected into 500µL 1×IMDM+20% FBS in a 12×75-mm polystyrene tube. Collected cells were then performed purity sort using the same gate strategy and sorted into 0.8 mL 1×IMDM+50% FBS in a 1.5 mL DNA LoBind tube.
Cells were cross-linked for 5 min with 1% formaldehyde at room temperature prior to quencing with 125mM glycin. Cross-linked cells were sonicated using Covaris S2 sonicator for 14 min with 5% duty, Intensity 3 and Bursts 200. The sonicated lysate was centrifuged at a speed of 14000×g for 10 min under 4°C. The cleared chromatin (10000 cells per 100 µl) in the supernatant was transferred to a new low-bind Eppendorf tube for subsequent microfluidic ChIP.
The ChIPed and input DNA was constructed into libraries by using the ThruPLEX FD Kit (Rubicon Genomics, Ann Arbor, MI, USA). During the amplification step, 11cycles was needed to get enough library from input DNA without over-amplification. 12~13 cycles is needed for ChIPed DNA extracted from 10000 cells. 14~15 cycles is normally required for ChIPed DNA from 1000 or less cells.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2500

Data processing

Raw sequencing reads were mapped to the human genome (hg19) or mouse genome (mm9) using Bowtie2 (v2.2.2) with default parameter setting. Only uniquly mapped reads were used for analysis
ChIP-seq peaks were identified by two peak caller, MACS (p-value < 10-5) and SPP (z-score > 4) with default parameter setting.
To generate the wig files, the human or mouse genome was divided into 100 bp bins, and the number of uniquely mapped reads were counted in each bin. We normalized the tag counts in each bin according to the total number of uniquely mapped reads. Input reads were processed in the same way and their normalized signals were subtracted from the ChIP-seq tracks.
Normalized wig files were converted to binary bigwig files
Genome_build: hg19 and mm9
Supplementary_files_format_and_content: bigwig files

Submission date

Feb 02, 2015

Last update date

May 15, 2019

Contact name

Kai Tan

Organization name

The University of Iowa

Department

Internal Medicine

Street address

3292 CBRB, 285 Newton Road