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| Status |
Public on Dec 23, 2019 |
| Title |
Human-PASseq-A1 |
| Sample type |
SRA |
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| Source name |
Testis
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Testis
developmental stage: Adult
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the human frozen testis tissues using mirVana miRNA isolation kit (Thermo Fisher, AM1560).
Briefly, we extracted poly(A)+ RNA from the total RNA using Dynabeads mRNA purification beads (Ambion, 61006). Poly(A)+ RNA was then fragmented at 70°C for 7 min by chemical hydrolysis yielding 60 to 80 nt fragments. Fragmented RNA was further reverse transcribed using barcoded oligo(dT) oligonucleotides containing sites for forward and reverse Illumina sequencing primers that were separated by a hexa-ethyleneglycol spacer (Sp18). The oligonucleotides contained unique molecular index (UMI), enabling the PCR duplicate removal, preceded by 5′p-GG for efficient cDNA circularization. cDNA was further circularized using Circligase ssDNA ligase (Illumina, CL4111K) at 60°C for 4 h. Following cDNA circularization, fourteen cycles of PCR amplification was performed using KAPA HiFi library amplification kit (Kapa Biosystems, KK2611).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
The 3′ adaptor sequence from the reads were removed and those reads whose PHRED score <5 was further filtered. The reads passed the quality check were mapped to human genome (hg19) and or mouse genome (mm10) using piPipes allowing 1 mismatch.
For RNA-seq, CAGE and PAS-seq, we first removed rRNA reads using bowtie 2.2.5 with default parameters48 and then the unaligned reads were further mapped to human genome (hg19) using STAR 2.3 (Ref. 52). Mapped results were generated as SAM format that was further transformed into duplication removed sorted BAM format using SAMtools 1.8 and normalized bigWig files are generated by custom scripts.
Raw reads were mapped to genome using Bowtie 2.2.5 with parameter —very-sensitive. Mapped results were generated as SAM format that was further transformed into duplication removed sorted BAM format using SAMtools 1.8. We, then, normalized genome coverage files in bigWig format that were calculated using homemade script. Data are presented in bigWig file of unique mapping reads as read depth per million reads (rpm). We used Model-based analysis of ChIP-seq (MACS 1.1.2; Ref. 58) with parameter -q 0.01 to detect A-MYB peaks that were significantly enriched over input (1,296 genomic regions; false discovery rate [FDR] < 0.05).
Genome_build: hg19; mm10
Supplementary_files_format_and_content: bigWig; narrowPeak; rpm.txt (text)
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| Submission date |
Aug 13, 2019 |
| Last update date |
Dec 23, 2019 |
| Contact name |
Tianxiong Yu |
| Organization name |
Umass Med
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| Street address |
364 Plantation St
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| City |
Worcester |
| State/province |
MA |
| ZIP/Postal code |
01605 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE135791 |
Pachytene piRNA Genes Are Rapidly Diverging Among Modern Humans |
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| Relations |
| BioSample |
SAMN10936567 |
| SRA |
SRX5376165 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4030237_TGACT.Dedup_Atrimmed.uniq.crick.bw |
25.1 Mb |
(ftp)(http) |
BW |
| GSM4030237_TGACT.Dedup_Atrimmed.uniq.watson.bw |
25.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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