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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 20, 2021 |
| Title |
RNAseq.mouse.testis.wt.pUPF1IP.adult.rep1 |
| Sample type |
SRA |
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| Source name |
testis
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| Organism |
Mus musculus |
| Characteristics |
tissue: whole testis
strain: C57B6
genotype: wt
age: adult
treatment: RIP
tissue: whole testis
molecule subtype: rRNA depleted RNA
rip antibody: anti-pUPF1 from Lynne Maquat's lab, Ser1127
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| Treatment protocol |
The mice were anesthetized with Ketamine/Xylazine mixture (Ketamine 100 mg/kg; Xylazine 25 mg/kg) via intraperitoneal injection. After complete anesthesia, testes were exteriorized with a longitudinal incision around 1 cm at the center of abdomen. The tunica albuginea was penetrated using a sharp 26 G needle (BD, Franklin Lakes, NJ, USA, 30511) 1 mm from the vascular pedicle, and the needle was withdrawn to generate path for introducing a blunt end Hamilton needle (Hamilton, Reno, NV, USA, 7786-02). PBS containing 0.02% Fast Green FCF (Thermo Fisher Scientific, Waltham, MA, USA) with 4EGI was slowly injected using a Hamilton microsyringe (1705RN) into one testis, and vehicle control without drug was injected into the other testis. The needle was held in place for 30 seconds before removal to prevent back flow of the solution. Successful completion of injection was indicated by testis filled with green solution. The testes were returned to the abdominal cavity after injection. The incisions were sutured. At the end point, the mice were euthanized by cervical dislocation, and the testes were collected. For immunoprecipitations, anti-CBP80 rabbit antibody (Bethyl laboratories, A301-794A) or anti-eIF4E rabbit antibody (Bethyl laboratories, A301-153A) were used.
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| Growth protocol |
Mice were maintained and used according to guidelines for animal care of the NIH and the University Committee on Animal Resources at the University of Rochester. White Leghorn testes of the Cornell Special C strain from one-year old roosters were used according to guidelines for animal care of the NIH and the University Committee on Animal Resources at the University of Rochester.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNAs were extracted using mirVana miRNA Isolation Kit (ThermoFisher, AM1560), and rRNAs were depleted from total RNAs with complementary DNA oligomers (IDT) and RNaseH (Invitrogen, Waltham, MA, USA)
1, Strand-specific RNA-seq: libraries were constructed following the TruSeq RNA sample preparation protocol. 2, Ribo-seq: Testes samples were lysed, RNase T1 & A treated and loaded on sucrose gradients, and 80S monosomes were recovered for library constrution. The 3'-adapter is TGGAATTCTCGGGTGCCAAGG. 3, Degradome: The RNAs were first oxidized at room temperature for 30 minutes with sodium periodate (Sigma, St. Louis, MO, USA) to block the 3´-ends from ligation, and were then size-selected to isolate RNA ≥ 200 nt (DNA Clean & Concentrator™-5, Zymo Research). 5´ adaptors were attached using T4 RNA ligase (Ambion) at 20°C for 3 hours. The ligated products were subjected to rRNA depletion with complementary DNA oligomers (IDT) and RNaseH (invitrogen). The rRNA depleted ligation products were reverse transcribed using a degenerate primer (5´-GCACCCGAGAATTCCANNNNNNNNC-3´). cDNA was amplified by PCR using KAPA HIFI Hotstart polymerase (Kapa Biosystems, Wilmington, MA, USA), and 250–350 nt dsDNA was isolated on 8% native PAGE gels. 4, Small RNAseq: Small RNA libraries were constructed and sequenced using oxidation to enrich for piRNAs by virtue of their 2´-O-methyl-modified 3´ termini. The oxidation procedure selects against in vitro-digested products with a 3´ phosphate. A 25-mer RNA with 2´-O-methyl-modified 3´ termini (5´-UUACAUAAGAUAUGAACGGAGCCCmG -3´) was used as a spike-in control. The 3'-adapter is TGGAATTCTCGGGTGCCAAGG.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
RNAseq
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| Data processing |
Analyses were performed using piPipes v1.4. All data from the small RNA sequencing, ribosome profiling (Ribo-seq), RNA sequencing, and degradome sequencing were analyzed using the latest mouse genome release mm10 (GCA_000001635.7), and chicken genome release galGal6 (GCA_000002315.5). Generally, one mismatch is allowed for genome mapping.
For small RNA sequencing, libraries were normalized to the sum of total miRNA reads. Uniquely mapping reads >23 nt were selected for further piRNA analysis. To ensure precision of the mapping, a piRNA is counted only when the 5´-end of the piRNA maps to the transcript.
For RNA-seq reads, the tpm (transcripts per million) values were quantified using the Salmon software.
For Ribo-seq analysis, uniquely mapping reads between 26 nt and 32 nt were selected for further analysis.
Genome_build: Mouse: mm10 (GCA_000001635.7), Chicken: galGal6 (GCA_000002315.5)
Supplementary_files_format_and_content: For Small RNAseq, processed data files are unique genome alignment results in BED2 format (https://github.com/bowhan/piPipes/wiki/smallRNA-seq). For Ribo-seq, results are unique genome alignment bed13 files, with an additional sequence column after a regular bed12 format. For Degradome, unique mapping reads are in bed12 format. RNAseq quantification results are .sf files output by Salmon software.
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| Submission date |
Oct 19, 2021 |
| Last update date |
Oct 20, 2021 |
| Contact name |
Xin Li |
| E-mail(s) |
Xin_Li@urmc.rochester.edu
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| Phone |
(585) 275-6576
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| Organization name |
University of Rochester
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| Department |
Biochemistry and Biophysics
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| Lab |
Li Lab
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| Street address |
601 Elmwood Ave
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| City |
Rochester |
| State/province |
NY |
| ZIP/Postal code |
14623 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE155350 |
Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs |
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| Relations |
| BioSample |
SAMN22416887 |
| SRA |
SRX12695709 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5640058_RNAseq.mouse.testis.wt.pUPF1IP.adult.rep1.sf.txt.gz |
767.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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