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| Status |
Public on Dec 23, 2019 |
| Title |
Human-ChIPseq-AMYB_input-A10 |
| Sample type |
SRA |
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| Source name |
Testis
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Testis
developmental stage: Adult
chip antibody: None
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total DNA was extracted from frozen tissues.
Briefly, frozen tissues were cut into small pieces on dry ice and transferred to 1.7 ml tubes containing cold PBS. Samples were then cross-linked with 2% (w/v) formaldehyde at room temperature for 30 min using end-over-end tumbler. Thereafter, fixed tissues were crushed in the presence of ChIP lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1) by 40 strokes with a “B” pestle of Dounce homogenizer (Kimble-Chase, Vineland, USA). Furthermore, lysed samples were sonicated using Covaris ultrasonicator (Covaris, E220) to shear the chromatin into 150 to 200 bp. Sonicated lysate was then diluted 1:10 with ChIP dilution buffer (0.01% SDS, 1.1% Triton X-1001.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl) and we performed immunoprecipitation using 5.5 µg of anti-A‑MYB (Sigma, HPA008791) and 4.5 µg of anti-H3K4me3 (Abcam, ab8580). Following the immunoprecipitation of the chromatin with the antibody of interest, we extracted DNA by phenol:chloroform:isoamyl alcohol (25:24:1) (pH 8) and prepared ChIP-seq libraries for anti-AMYB, anti-H3K4me3 and input DNA as previously described.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| 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 |
SAMN10936576 |
| SRA |
SRX5376195 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4030246_6615NT-AMYB-input-Id3_S12.rpm.uniq.bw |
45.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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