|
| Status |
Public on Oct 24, 2016 |
| Title |
gDNA-Input 2 |
| Sample type |
SRA |
| |
|
| Source name |
Kidney podocyte
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Kidney podocyte
immortilization: SV40-Temperature Sensitive T-Antigen Immortalized
passage: 10-12
|
| Treatment protocol |
n/a
|
| Growth protocol |
Temperature Sensitive SV40-T antigen Immortalized podocytes were cultured first at 33 degrees in RPMI-1040 supplemented with 10% FBS and 1% Antibiotics, cells were thermoswitched to 37 degrees to induce differentiation and cultured for 10 days in DMEM (1g glucose/L) supplemented with 5% FBS and 1% Antibiotics.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Gluteraldehyde crosslinked cells were subjected to nuclear extraction protocol and nuclei were subjected to sonication by BioRuptor. 1% Input Chromatin was set aside and Chromatin was incubated with biotynilated Tug1-Even or Tug1-Odd oligos, DNA was reverse-crosslinked and purified by ethanol precipitation.
10 ng of DNA samples were prepared for Illumina sequencing as the following steps: 1) DNA samples were blunt-ended with T4 DNA polymerase and Klenow polymerase; 2) a dA base was added to the 3' end of each strand by Klenow (exo minus) polymerase; 3) Illumina's genomic adapters were ligated to the DNA fragments; 4) PCR amplification was performed to enrich ligated fragments; 5) Enriched product of ~200-400bp was cut out from gel and purified with QIAquick Gel Extraction Kit. The completed libraries were quantified by Agilent 2100 Bioanalyzer. The libraries were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cell, amplified in situ. The libraries were then sequenced on the Illumina HiSeq 2000 following the TruSeq Rapid SBS Kit protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
ChIRP-Seq
|
| Data processing |
After the sequencing platform generated the sequencing images, the stages of image analysis and base calling were performed using Off-Line Basecaller software (OLB V1.8).
After passing Solexa CHASTITY quality filter, the clean reads were aligned to mouse genome (UCSC mm10) using BOWTIE software (V2.1.0).
peak calling of the ChIRP regions using MACS V1.4.0. Statistically significant ChIRP-enriched regions (peaks) were identified by comparison to input, using a p-value threshold of 10-5.
The peaks in samples were annotated by the nearest gene using the newest UCSC RefSeq database
Genome_build: mm10
Supplementary_files_format_and_content: bed
|
| |
|
| Submission date |
Feb 02, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Shawn Samson Badal |
| Organization name |
MD Anderson Cancer Center
|
| Department |
Nephrology
|
| Lab |
Danesh
|
| Street address |
2121 W. Holcombe Blvd
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE77493 |
Genome wide map of long noncoding RNA Tug1 binding sites in cultured mouse podocytes |
|
| Relations |
| BioSample |
SAMN04452924 |
| SRA |
SRX1557613 |
