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Status |
Public on Aug 03, 2022 |
Title |
ChIP Input H3K27ac Interzone Rep1 |
Sample type |
SRA |
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Source name |
ChIP Input H3K27ac Interzone
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Organism |
Gallus gallus |
Characteristics |
developmental stage: HH32 tissue: interzone chip antibody: none
|
Growth protocol |
Chick White Leghorn embryonated eggs were incubated at 38.5ÂșC in fixed humidity for 7.5 days, followed by evaluation of developmental stage based on the Hamilton Hamburger classification (HH). The joint interzones and adjacent phalange samples were microdissected from hindlimb digit-3
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Extracted molecule |
genomic DNA |
Extraction protocol |
For the preparation of ChIPseq libraries, the histone-DNA complexes were immunoprecipitated with antibody. For each sample, 100 specimens were collected from the 3rd hindlimb digit of developing chicken ebmryo (HH32) and pooled together. Further, samples were dissociated using Collagenase-II and passed through a 40-m cell strainer. The 1mln of cells were cross-linked and after series of washes the cell nuclei were further isolated. Subsequently, the nuclei were treated with SDS-containing lysis buffer, and chromatin was sheard obtaining the average size of 150 bp. For ChIP, 500 ng of sonicated chromatin was immunoprecipitated with 7.5 g of antibody. Input sample was collected prior to immunoprecipitation reaction. ChIP-seq libraries were prepared using QIAseq Ultra Low Input Library Kit (QIAGEN, Hilden, Germany). Briefly, DNA was end-repaired, adenosines were added to the 3 ends of dsDNA and adapters were ligated. Adapters containing DNA fragments were amplified by PCR using NEB starters. Mean library size was 300 bp. Libraries were run in the rapid run flow cell and were single-end sequenced (65 bp) on HiSeq 1500 (Illumina, San Diego, CA 92122 USA).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 1500 |
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|
Description |
Interzone_H3K27ac_IP_rep1_macs2_broad_peaks.broadPeak
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Data processing |
In the ChIPseq primary analysis, the quality of reads was checked by FASTQC followed by removal of adapters using Trimmomatic tool. Reads were mapped to the Gallus Gallus 6 reference genome using Bowtie2 followed by removal of PCR duplicates with Picard tool. Peaks were called with MACS2. In the RNAseq primary analysis, the quality of reads was validated with FASTQC and adapters were removed using Trimmomatic tool. Reads were aligned to the Gallus Gallus 6 reference genome using using STAR Reads were counted by gene using FeatureCounts Identification of differentially expressed genes was performed using DESeq2 Assembly: gg6 Supplementary files format and content: (RNA-seq) DESeq2 normalized abundance measurements ouput Supplementary files format and content: (ChIP-seq) MACS2 narrow peaks files
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Submission date |
Mar 16, 2022 |
Last update date |
Aug 03, 2022 |
Contact name |
Karol Nowosad |
E-mail(s) |
karolnowosad.lab@gmail.com
|
Organization name |
Erasmus MC
|
Street address |
Doctor Molewaterplein 40
|
City |
Rotterdam |
ZIP/Postal code |
3015 GD |
Country |
Netherlands |
|
|
Platform ID |
GPL21476 |
Series (1) |
GSE198819 |
Identification of candidate regulatory elements controlling transcriptome during the formation of interphalangeal joints |
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Relations |
BioSample |
SAMN26733781 |
SRA |
SRX14490266 |