|
| Status |
Public on Oct 16, 2014 |
| Title |
ChIP-N_WT |
| Sample type |
SRA |
| |
|
| Source name |
thymocytes, WT, Zfp335/NIF1 ChIP
|
| Organism |
Mus musculus |
| Characteristics |
strain/background: C57BL/6
genotype/variation: WT
cell type: total thymocytes
chip antibody: Zfp335/NIF1 (A300-797A, Bethyl Laboratories, lot no. A300-797A-1)
|
| Treatment protocol |
NA
|
| Growth protocol |
Total thymocytes were isolated by mechanical disaggregation through a 40μm nylon sieve and washed in cold PBS before formaldehyde crosslinking for ChIP.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sonicated nuclei were cleared by centrifugation and incubated overnight with anti-Zfp335 bound to Dynabeads. Beads were washed in low-salt buffer, high-salt buffer, LiCl buffer and TE buffer. Protein/DNA complexes were eluted, reverse-crosslinked overnight at 65°C and treated with RNase A and proteinase K. ChIP DNA was purified using QIAquick PCR Purification kit (Qiagen).
7-10 ng ChIP DNA and 10 ng input DNA were used for library preparation, performed according to Illumina's TruSeq protocol with some modifications. DNA clean-up, removal of adapter dimers and size selection (300-500 bp) were done using Agencourt AMPure XP beads (Beckman Coulter). Libraries were checked for quality using the High Sensitivity DNA Bioanalyzer kit (Agilent Technologies), quantified with the Qubit dsDNA HS Assay kit (Life Technologies), and sequenced as 50 bp single-end reads on the Illumina HiSeq 2000 platform.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
A300-797A Zfp335 ChIP, WT
|
| Data processing |
Base calling was performed using RTA (Real-Time Analysis) 1.13.
Illumina adapter sequences were trimmed from reads using cutadapt 1.4.1. Trimmed reads were aligned to the mm9 genome assembly using bwa 0.7.7, allowing for a maximum of 2 mismatches.
Reads aligned with a MAPQ score of less than 20 were filtered out using samtools.
Peaks were called using MACS2 (parameters: -g mm --bw=300 –q 0.05).
Aligned sequence reads were filtered with samtools rmdup and used for generating normalized pileup tracks with MACS2 callpeak --SPMR (fragment pileup per million reads), which were then converted to the bigWig format.
Genome_build: MGSCv37 (mm9)
Supplementary_files_format_and_content: bigWig: normalized signal tracks; narrowPeak: peaks called by MACS2.
|
| |
|
| Submission date |
Jun 09, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Brenda Yuyuan Han |
| Organization name |
Institute of Molecular and Cell Biology, A*STAR
|
| Street address |
61 Biopolis Drive, Proteos
|
| City |
Singapore |
| ZIP/Postal code |
138673 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE58293 |
Zinc finger protein Zfp335 is required for formation of the naïve T cell compartment |
| GSE58333 |
Genome-wide profiling of Zfp335 binding sites in thymocytes |
|
| Relations |
| BioSample |
SAMN02848481 |
| SRA |
SRX583296 |
