|
| Status |
Public on Sep 19, 2019 |
| Title |
Cnt - pPPR explants H3K27ac_ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
Cnt - pPPR explants cultured for 6hrs
|
| Organism |
Gallus gallus |
| Characteristics |
developemntal stage: HH6 + 6hrs culture
culture condition: DMEM
chip antibody: anti-H3K27ac - Abcam - ab4729 (1 ug/ul Rabbit)
|
| Treatment protocol |
Expants were supplemented with FGF2 (0.25ng/ul; R&D)
|
| Growth protocol |
Fertilized chicken eggs incubated in a humidified incubator at 38°C until reached HH6 (0 somites), embryos were collected in Tyrode’s saline and progenitors (pPPR) were dissected using fine needles, and cultured in DMEM for 6hrs
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Explants were dissociated in 0.5ml of Nuclei Extraction Buffer, homogenized and cross-linked using 1% formaldehyde for 9 min at room temprature, 0.125M glycine final concentration was used to stop fixation. Total of 100 tissues were pulled together and sonicated.
Chromatin was amplified with a step of linear amplification (15 cycles) following the protocol described in (Adli and Bernstein, 2011). Libraries were prepared according to nano-ChIP-seq (Adli and Bernstein, 2011) with the exception that only 12 cycles were used in the PCR amplification, following Illumina's instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Basecalls performed using Sanger/Illumina 1.9
Trimming was carried out at the 3' end when sequence quality was poor (Phred score<10) Trimmed reads were then aligned to the chick genome Galgal4.71 using Novoalign (Novocraft 2.08.01)
Peak calling was carried out using Homer (FC>1.5 relative to input; FDR 0.01) and MACS (p-value 1e-5; FDR 0.05)
Putative enhancers were identified in the following way: regions of up to 3 kb flanked by H3K27ac and devoid of H3K27me3 peaks were identified and assigned to the nearest gene using gene annotations from Ensembl (Galgal4.71) and refGene (Nov. 2011 ICGSC Gallus_gallus-4.0/galGal4).
Common and unique putative enhancers for +FGF2-treated and control samples were identified using the R package ChIPpeakAnno
Genome_build: gg4
|
| |
|
| Submission date |
Sep 18, 2019 |
| Last update date |
Sep 19, 2019 |
| Contact name |
Andrea Streit |
| E-mail(s) |
andrea.streit@kcl.ac.uk
|
| Organization name |
Kings College London
|
| Department |
Centre for Craniofacial & Regenerative Biology
|
| Lab |
Streit
|
| Street address |
Centre for Craniofacial & Regenerative Biology, King's College London
|
| City |
London |
| ZIP/Postal code |
SE1 9RT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10223 |
| Series (1) |
| GSE137664 |
Enhancer activation by FGF signalling during otic induction |
|
| Relations |
| BioSample |
SAMN12783434 |
| SRA |
SRX6867512 |
