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Sample GSM1678004 Query DataSets for GSM1678004
Status Public on May 08, 2015
Title Chicken-DT40-WT
Sample type SRA
 
Source name DT40-Chicken cell line-mutated WT
Organism Gallus gallus
Characteristics cell line: DT40-WT
Extracted molecule other
Extraction protocol Nuclei were extracted from tissues and cell lines. All the reagents used were pre-chilled and the entire procedure was performed on ice. Tissues and cells were homogenized by douncing 50 times in 10 mL of Nuclear Extraction Buffer. After homogenization, samples were layered on 18 mL of Sucrose Cushion in 40 mL ultracentrifuge tubes (Beckman, 25 × 89 mm, 344058) and spun at 30,000 x g for 45 min at 4°C (Beckman, L8-70M, SW28 rotor). Supernatant, including debris, was removed, and 200 μl of chilled 1 × PBS was added to the tube. After 20 min incubation on ice, the nuclei were dissociated by pipetting, and recovered by centrifugation at 500 x g for 5 min.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina Genome Analyzer IIx
 
Description Circular DNA
Extrachromosomal circular DNA
Data processing Illumina Casava1.7 software used for basecalling.
Identification of microDNA by paired-end sequencing of MDA products: microDNA were computationally identified by two different methods: Island method and split read method (in-house developed). All sequence tags were mapped on the reference genome (build mm10 for mouse and hg19 for human galGal4 for chicken) by using the Novoalign software. Only those paired tags that could be mapped uniquely and in opposite orientation to each other were considered for the identification of islands in the "island method". The sequence coverage of each base pair was profiled for each chromosome, and islands (potential circles) delineated where there were two consecutive sequenced bases. In other words an "island" is a contiguous portion of the genome where a cluster of PE-tags were mapped uniquely and in the opposite orientation to its pair. Paired End tags that had one tag mapping uniquely to an island, while the other was unmapped but passed the sequence quality filter, were considered separately for the validation of circles. We generated a database of hypothetical junctional tags that could be created by ligation of the ends of islands to generate circles. The unmapped tags described above were matched against these hypothetical junctional tags. If the mapped tag of a PE read falls in an island and the un-mapped tag matches with a hypothetical junctional tag generated by ligation of the ends of the island, we annotated that island as a circle. This approach underestimates the number of true microDNAs because further sequence coverage is likely to reveal more junctional tags ligating the ends of islands. The "split read" method extracted the paired end tags where one end was mapped (M) and other end was un-mapped (UM) but had good base quality score. The UM tags were split into two equal halves and mapped individually by using Novoalign software. Those split pairs which were mapped on the same strand (F-F or R-R) with no mismatch were considered for the identification of circles. The "Island method" and the "split-read method" both are dependent on the junctional tags for the identification of circles. >80% of the circles were identified by both algorithms. The "Island method" failed to detect circles 1) with low base coverage, 2) with the start and the end of the circle contaminated by reads from contaminating linear DNA or lost due to sequencing errors and 3) that were overlapping with each other. On the other hand the "Split-read method" failed to detect circles if the two split reads do not map uniquely to the genome. Processed data include the microDNA co-ordinate (chromosome name, start position of circle and end position of circle) mm9 assembly for mouse and hg19 assembly for human samples. The fourth column in the processed data is base pair coverage (abundance).
Genome_build: mm10 for mouse and hg19 for human galGal4 for chicken
Supplementary_files_format_and_content: Processed data include the microDNA co-ordinate (chromosome name, start position of circle and end position of circle) mm10 assembly for mouse, hg19 assembly for human and galGal4 for chicken samples. The fourth column in the processed data is base pair coverage (abundance).
 
Submission date May 07, 2015
Last update date May 15, 2019
Contact name Anindya Dutta
E-mail(s) ad8q@virginia.edu, pk7z@virginia.edu
Phone 434 - 924 - 2466
Organization name University of Virginia
Department MD-BIOC BC/Mole Genetics~School of medicine
Lab Anindya dutta lab
Street address Room no. 1232, Jordan Hall
City Charlottesville,
State/province VA
ZIP/Postal code 22903
Country USA
 
Platform ID GPL13797
Series (1)
GSE68644 Extrachromosomal DNA (microDNA) in Human, Mouse and Chicken
Relations
BioSample SAMN03611439
SRA SRX1020699

Supplementary file Size Download File type/resource
GSM1678004_DT40-WT.microDNA.txt.gz 937.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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