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| Status |
Public on Jul 30, 2021 |
| Title |
FH-Piwi-IP_1_3 |
| Sample type |
SRA |
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| Source name |
OSC cell line
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: ovarian somatic sheet cells (OSC)
genotype: 3xFH-Piwi / WT
biological_replicate: 1
technical_replicate: 3
ip antibody: anti-Flag M2 magnetic beads, Millipore Sigma, M8823
5`_adapter: GTTCAGAGTTCTACAGTCCGACGATCNNNNNNNN
3`_adapter: NNGCGATATGGAATTCTCGGGTGCCAAGG
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| Growth protocol |
OSC cell culture was grown as described in protocols described here: https://dgrc.bio.indiana.edu/product/View?product=288
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| Extracted molecule |
total RNA |
| Extraction protocol |
piRNAs: RNA was extracted from the OSC cell lysate by immunoprecipitating PIWI proteins using anti-Flag antibody beads or custom made fly anti-Piwi antibodies. Total small RNAs: RNA was extracted from the cells usign PureLink miRNA Isolation kit (Thermo Fisher Scientific, K157001)
piRNA libraries: Imunoprecipitated RNA was ligated to 3` adapter containing unique indexes and two random bases (2N), and ligation products in 48 to 58-nt size range (equivalent to 19 - 29-nt long initial fragments) were recovered from Urea PAGE gel. Purified fragments were then ligated to 5` adapter containing eight random bases (8N) and purified on Urea PAGE gel again by selecting fragments in 82 - 92-nt size range. Final fragments were PCR amplified to add sequencing adapters. Total small RNAs libraries: A pellet of one million cells was used to purifiy small RNAs using PureLink miRNA Isolation kit (Thermo Fisher Scientific, K157001) and were spiked with a mix of 26 nt-long synthetic RNA callibrator sequences. RNA was ligated to 3` adapter containing unique indexes and two random bases (2N), and ligation products in 48 to 58-nt size range (equivalent to 19 - 29-nt long initial fragments) were recovered from Urea PAGE gel. Purified fragments were then ligated to 5` adapter containing eight random bases (8N) and PCR ampliffied to add sequencing adapters.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
Piwi-associated RNA (piRNA)
size-selected piRNAs with 10 UMIs
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| Data processing |
Fastq files were trimmed using cutadapt (v2.3) to remove the 5` and 3` adapters and keep 10 unique molecular identifiers (UMI) - 8N's at 5` end and 2N's at 3` end.
Fastq files were processed in R using custom script where sequences were collapsed by UMI to remove PCR duplicates, UMI's were trimmed, and sequences shorter than 18-nt in length were removed. To reduce the file size while retaining all the information, the sequences were collapsed and individual sequence abundance was recorded in fasta header. Fasta header is comprised of sample name that is followed by a unique alphanumeric string containing sequence ID number (S) and abundance count (M) and have following structure: SAMPLE_XY - S[UniqueSequenceID]M[SequenceAbundance]. Fasta header "FH-Piwi-IP_1_1-S32M12" can be split into sample name ("FH-Piwi-IP_1_1") and unique string ("S32M12") that can be further split into unique sequence ID ("S32") and abundance count of this particular sequence ("M12"). Please see UNIQSEQS.fa processed files. These files are deposited for the convenient, fast download and analysis.
UNIQSEQS.fasta files were aligned to genome comprised of the structural RNAs (tRNA, rRNA, snRNA, snoRNA) using STAR (v2.5.2b), the unmapped sequences were collected and mapped to the dm6 genome using the same aligner. The obtained .bam files were analyzed in R using custom scripts.
piRNA quantification was performed by determining the number of cells per sample (S1-S8) using molecular counts of the callibrator oligoes in the UNIQSEQS.fasta files. MiRNAs were quantified by mapping to miR Base (Release 22.1). PiRNAs were quantified by considering seqeunces in 24 - 29-nt size range.
Genome_build: dm6 (Drosophila melanogaster)
Supplementary_files_format_and_content: xlsx: piRNA_quantification; fasta: custom trimmed and filtered unique sequence files
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| Submission date |
Aug 11, 2020 |
| Last update date |
Jul 30, 2021 |
| Contact name |
Pavol Genzor |
| E-mail(s) |
[email protected]
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| Organization name |
Henry M Jackson Foundation
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| Department |
ACESO
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| Street address |
6720A Rockledge Drive, Suite 100
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20817 |
| Country |
USA |
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| Platform ID |
GPL23323 |
| Series (1) |
| GSE156058 |
Cellular abundance shapes function in piRNA-guided genome defense |
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| Relations |
| BioSample |
SAMN15792768 |
| SRA |
SRX8926649 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4721508_FH-Piwi-IP_1_3.UNIQSEQS.fa.gz |
62.3 Mb |
(ftp)(http) |
FA |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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