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| Status |
Public on May 24, 2021 |
| Title |
SSY-Karenina-CENPA |
| Sample type |
SRA |
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| Source name |
Siamang (Symphalangus syndactylus)
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| Organism |
Symphalangus syndactylus |
| Characteristics |
cell line: EBV-tranformed lymphocyte cell line
chip antibody: CENP-A (ab13939)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We performed ChIP-seq using the Magnify ChIP kit (Thermo Fisher Scientific) according to manufacturer’s instructions with minor modifications. Briefly, per ChIP assay we fixed 3x105 EBV transformed gibbon LCLs with 1% formaldehyde at room temperature for 10 min. Fixation was quenched with glycine, and cells were washed three times with cold 1XPBS. Cells were lysed for 10 min on ice using the Magnify Lysis buffer, in presence of Proteinase inhibitor cocktail. Lysed cells were then sonicated using the Bioruptor Pico sonicator (Diagenode) for 12 cycles (30 sec on/off) and spun down to remove cell debris. A 1% aliquot was taken from the chromatin as input, and the rest was incubated with the appropriate antibody [CENP-A (ab13939): 2ug, CENP-B (ab25734): 4ug, CENP-C (ab50974): 2ug] at 4℃, with rotation overnight. The next day we incubated samples with Dynabead protein A/G rotating at 4℃ for 2 hours, followed by bead washes, reverse-crosslink and DNA purification according to Magnify ChIP kit protocol.
All sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) for 1-5ng of starting material and without size selection. The Qubit dsDNA High Sensitivity kit (Thermo Fisher Scientific) and Agilent Bioanalyzer 2100 were used to QC the libraries.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
CENP_BroadPeaks_sortedbyFE.xlsx
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| Data processing |
DATA PROCESSING STEPS TO GENERATE THE *summary.txt FILES:
Raw reads were trimmed using Trimmomatic [11] to remove adaptor sequences, as well as low quality and short reads (2:30:10:26:true SLIDINGWINDOW:4:20 TRAILING:20 LEADING:20 MINLEN:50).
We used seqtk to randomly select 2.5 million trimmed read pairs from each ChIP sample. The random subsets of reads were converted to fasta format using manual bash scripts.
Repeats in the pre-processed ChIP-seq libraries were annotated using RepeatMasker version 4.0.3. The gibbon (Hylobates sp.) 20150807 Repbase library was used for annotation.
We used a custom script to group repeats in each library based on repeat class and family in each of the read pair files, and to calculate the %count and %length (of total repeats) they constituted
Genome_build: Not applicable
Supplementary_files_format_and_content: Text files summarizing the %count and %length of each repeat, repeat family and repeat class among randomly subsampled reads from each ChIP library.
DATA PROCESSING STEPS TO GENERATE THE *contigs.fa FILES:
We used the K-mer Analysis Toolkit (KAT) to determine the optimal k-mer size and predicted genome coverage for each library.
We used RepARK for de novo assembly of reads from each ChIP-seq library.
Genome_build: not applicable
Supplementary_files_format_and_content: Fasta files representing assembled contigs obtained from each ChIP-seq library.
DATA PROCESSING STEPS TO GENERATE THE CENP_BroadPeaks_sortedbyFE.xlsx FILE:
Raw reads were trimmed using Trimmomatic to remove adaptor sequences, as well as low quality and short reads (2:30:10:26:true SLIDINGWINDOW:4:20 TRAILING:20 LEADING:20 MINLEN:50).
Trimmed reads were aligned to the gibbon genome (Nleu3.0) using Bowtie2, with "very sensitive" paired-end settings.
Significant (q<0.01) broad peaks were identified using MACS2,while allowing maximum fof two read duplcates. Peaks were sorted based on fold enrichment.
Genome_build: Nleu3.0
Supplementary_files_format_and_content.xlsx spreadsheet containing the coordinates of all broad peaks across CENP libraries. Peaks are sorted from high fold-enrichment (FE) to low FE.
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| Submission date |
Nov 11, 2020 |
| Last update date |
May 25, 2021 |
| Contact name |
Mariam Okhovat |
| Organization name |
Oregon Health and Science University
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| Department |
Medicine
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| Street address |
3030 S Moody Ave.
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| City |
Portland |
| State/province |
Oregon |
| ZIP/Postal code |
97239 |
| Country |
USA |
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| Platform ID |
GPL29393 |
| Series (2) |
| GSE161217 |
Comparative analyses of gibbon centromeres reveal dynamic species-specific shifts in repeat composition |
| GSE161308 |
Comparative analyses of gibbon centromeres reveal dynamic species-specific shifts in repeat composition [ChIP-seq] |
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| Relations |
| BioSample |
SAMN16769180 |
| SRA |
SRX9487811 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4904653_Karenina-CENPA-1P.oneil-pipeline.all.reps.out_summary.txt.gz |
56.2 Kb |
(ftp)(http) |
TXT |
| GSM4904653_Karenina-CENPA-2P.oneil-pipeline.all.reps.out_summary.txt.gz |
56.5 Kb |
(ftp)(http) |
TXT |
| GSM4904653_kareninacenpa_k37_t30_contigs.fa.gz |
195.4 Kb |
(ftp)(http) |
FA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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