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| Status |
Public on Nov 30, 2015 |
| Title |
Sample4_NPC_p53-Sox2 |
| Sample type |
SRA |
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| Source name |
iPSC-derived human Neural Progenitor Cell with P53 knockdown
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: Sox2
cell type: Neural Progenitor Cell
treatment: Inhibition of P53
cell line: iPSC derived
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| Growth protocol |
Neurobasal media (Invitrogen) supplemented with N2 (Invitrogen), B27 (Invitrogen), and FGF2 (10 ng/ml; preprotech)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3*105 cells were fixed using 1% PFA, treated with glycine 0.1M and resuspended in nuclei lysis buffer (SDS 1%, EDTA 100mM, Tris-HCl 50mM pH7.5). Chromatin samples were sonicated with a Branson Digital Sonifier to generate DNA fragments from 200 to 1000 bp, diluted with ChIP RIPA Buffer (Tris-HCl 0.1mM pH7.5, EDTA 1mM, EGTA 0.5mM, Triton X-100 1%, SDS 0.1%, Deoxycolic acid 0.1%, NaCl 140mM) and incubated with Dynabeads Protein A (Invitrogen) coupled to the specific antibody. After overnight incubation, the immunocomplexes were eluted in ChIP elution buffer (Tris-HCl 20mM pH7.5, EDTA 5mM, NaCl 140mM, SDS 1%, proteinase K 40mg/ml) and the crosslinking was reverted overnight at 65ºC. DNA was recovered by phenol/chloroform extraction and quantified by quantitative PCR using SYBR green on an ABI Prism 7300 system.
ChIP DNA was prepared into ChIP-Seq libraries using NEBNext Ultra DNA Library Prep Kit for Illumina according to manufacturer's instructions except adapters were purchased from Bioo Scientific. Briefly, ChIP DNA was end-repaired, adenylated and adapter ligated. Adapter-ligated DNA was subjected to 12 cycles of PCR using NEB Q5 PCR mastermix. AMPure beads were used to purify sequencing libraries from reactions (Beckman Coulter). ChIP-Seq libraries were quantified using Invitrogen Qubit, Agilent Tape station and Kapa library quantification qPCR kit. Libraries were pooled and sequenced single-end 50bp on Illumina HiSeq 2500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Sancho_Martinez_et_al_ChIP_Sox2.txt
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| Data processing |
HiSeq Control Software (HCS v2.2.38) and Real Time Analysis (RTA v1.18.61) were used for base calling. Demultiplexing and BCL files conversion to FASTQ files were done by CASAVA v1.8.2.
Fastq files were mapped using bowtie2 v. 2.2.3 and deduplicated using Samtools 0.1.19.
Mapped reads were loaded into Seqmonk (v. v0.27.0) and duplicate reads were removed. Peaks were defined as a contiguous set of reads that were enriched by at least ten-fold. A 50 base pair gap was allowed to prevent large peaks from being broken into multiple small peaks. The number of reads in each peak were counted, corrected for total read count and log transformed.
Genome_build: hg19
Supplementary_files_format_and_content: Text files of peak locations by chromosome and start and stop sites. The sample columns indicate the number of reads in each peak which were counted (corrected for total read count and log transformed).
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| Submission date |
Mar 25, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert E Morey |
| E-mail(s) |
robmoreyucsd@gmail.com, remorey@health.ucsd.edu
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| Organization name |
UCSD
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| Department |
Pathology
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| Lab |
Parast
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| Street address |
2880 Torrey Pines Scenic Dr
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE67282 |
Establishment of tractable human iPSC-based models for the study and targeting of glioma initiation (ChIP-Seq) |
| GSE67286 |
Establishment of human iPSC-based models for the study and targeting of glioma initiating cells |
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| Relations |
| BioSample |
SAMN03447187 |
| SRA |
SRX968911 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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