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Sample GSM4030236 Query DataSets for GSM4030236
Status Public on Dec 23, 2019
Title Human-CAGEseq-J1-rep2
Sample type SRA
Source name Testis
Organism Homo sapiens
Characteristics tissue: Testis
developmental stage: Juvenile
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, following the rRNA removal, poly(A)+ RNA was extracted from the total RNA using Dynabeads mRNA purification beads (Ambion, 61006). RNA samples were heated to 68°C for 5 min and chilled on ice immediately. To prevent the 5′-end phosphorylated RNA entering to the library, we treated RNA samples with ten units of Antarctic Phosphatase (NEB, M0289) at 37°C for 1h. After heat inactivating Antarctic Phosphatase at 70°C for 5 min, we extracted RNA by phenol:chloroform:isoamyl alcohol (25:24:1; pH 6.7). Thereafter, 5′-end cap was removed from the RNA samples using twenty units of Tobacco Decapping Enzyme (Enzymax, LLC, 87) in the presence of 1 mM MnCl2 at 37°C for 2 h followed by phenol:chloroform:isoamyl alcohol extraction. After 5′ ligation, we performed reverse transcription using random oligo primer containing 3′ adaptor sequence. We then performed two cycle of PCR and purified the PCR products 200 to 400 bp. After that we perform ten more cycle of final PCR.
Library strategy RNA-Seq
Library source transcriptomic
Library selection CAGE
Instrument model Illumina NextSeq 500
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)
Submission date Aug 13, 2019
Last update date Dec 23, 2019
Contact name Tianxiong Yu
Organization name Umass Med
Street address 364 Plantation St
City Worcester
State/province MA
ZIP/Postal code 01605
Country USA
Platform ID GPL18573
Series (1)
GSE135791 Pachytene piRNA Genes Are Rapidly Diverging Among Modern Humans
BioSample SAMN10936584
SRA SRX5376166

Supplementary file Size Download File type/resource 9.7 Mb (ftp)(http) BW 9.7 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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