|
| Status |
Public on Sep 13, 2012 |
| Title |
Chicken_st11-14_NCC_H3K27ac |
| Sample type |
SRA |
| |
|
| Source name |
stage11-14_NCC_H3K27ac_ChIP
|
| Organism |
Gallus gallus |
| Characteristics |
tissue source: stage 11-14 chicken embryos
cell type: Neural crest cells (NCC)
chip antibody: Anti-Histone H3 (acetyl K27) antibody
chip antibody cat. #: Abcam
chip antibody vendor: ab4729
|
| Treatment protocol |
Chicken stage 11-14 NCC: Chick embryos were harvested at st.8 and the dorsal neural tube was excised and plated on matrigel coated plates in NCC differentiation media: 1:1 Neurobasal medium/D-MEM F-12 medium (Invitrogen), 0.5x B-27 supplement with Vitamin A (50x stock, Invitrogen), 0.5x N-2 supplement (100x stock, Invitrogen), 20 ng/ml bFGF (Peprotech), 20 ng/ml EGF (Sigma-Aldrich), 5 µg/ml bovine insulin (Sigma-Aldrich) and 1x Glutamax-I supplement (100x stock, Invitrogen).
|
| Growth protocol |
Chicken stage 11-14 NCC: Chick embryos were harvested at st.8 and the dorsal neural tube was excised and plated on matrigel coated plates. After between 48-72 hours cranial neural crest cells began to migrate out of the dorsal neural tube explants and considered to resemble st11-14 NCC from embryos. Cells were then trypsinized an pooled for ChIP assays. Chicken stage 20 NCC: Frontonasal prominences (FNP) NCCs were isolated in cold PBS at St20 according to Hamburger-Hamilton criteria. FNPs were placed in 1.26 U dispase in 1x PBS for 15 minutes at room temperature, then into DMEM with 10% FBS. Surface ectoderm and forebrain neuroectoderm were removed using sharpened tungsten needles leaving only FNP NCCs. Cells were then trypsinized an pooled for ChIP assays.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and ChIP-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA was size selected on agarose gel from 200-700 bp. Then isolated DNA was PCR amplified with Illumina primers for 18 cycles. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
ChIP against H3K27ac
|
| Data processing |
Enrichment analysis was performed using QuEST software with suggested settings for transcription factor binding.
Genome_build: galGal3
Supplementary_files_format_and_content: aligned.bed and calls.bed files including aligned reads and annotated peaks/regions are provided respectively
|
| |
|
| Submission date |
May 21, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Alvaro Rada |
| E-mail(s) |
alvaror@stanford.edu
|
| Organization name |
Stanford University
|
| Department |
Chemical and Systems Biology
|
| Lab |
Wysocka lab
|
| Street address |
269 Campus Drive, CCSR Building
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL13797 |
| Series (1) |
| GSE38066 |
Epigenomic profiling of enhancers identifies orphan nuclear receptors NR2F1 and NR2F2 as novel regulators of human neural crest. |
|
| Relations |
| SRA |
SRX148741 |
| BioSample |
SAMN00996668 |
