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| Status |
Public on Aug 30, 2019 |
| Title |
Chr18-/- ssRNA-seq (HC3802-7) |
| Sample type |
SRA |
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| Source name |
KO Testis
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| Organism |
Mus musculus |
| Characteristics |
genotype: KO
strain: B6D2F1
age: post natal day 28
tissue: testis
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| Treatment protocol |
Isolated testes total RNA was incubated with 5X borate buffer (148 mM borax, 148 mM boric acid, pH 8.6, Thermo Fisher Scientific), 10 min at room temperature for periodate reaction and β-elimination as well as freshly dissolved 200 mM NaIO4 (Thermo Fisher Scientific). To quench unreacted NaIO4, glycerol (ThermoFisher Scientific) was added and incubated for 10 min at room temperature. After adding 1X borate buffer, RNA was precipitated with ethanol for 1 hr, at -80 °C. After centrifugation, the RNA was dissolved in 1X borax buffer (30 mM borax and 30mM boric acid, 17.5 mM NaOH, pH 9.5) and incubated for 90 min at 45 °C prior to addition 1X borate buffer and glycogen. The RNA was precipitated with ethanol for 1 hr, at -80 °C, collected by centrifugation and dissolved in water. During β-elimination, periodate-reacted RNAs were shortened by 1 nt at the 3’ end with monophosphates and were unable to be amplified during library preparation. piRNAs, protected from β-elimination by 2’-O-methylation at 3’ end, were enriched in the non-coding RNA-seq libraries.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from testes samples was isolated using miRNAeasy Mini (Qiagen). The sequencing libraries were constructed from 100 ng – 1,000 ng of total RNA using the Illumina’s TruSeq Stranded Total RNA kit with Ribo-Zero following the manufacturer instruction. The fragment size of RNAseq libraries was verified using the Agilent 2100 Bioanalyzer (Agilent) and the concentrations were determined using Qubit instrument (LifeTech). The libraries were loaded onto the Illumina HiSeq 3000 for 2x50 bp paired end read sequencing. The fastq files were generated using the bcl2fastq software for further analysis.
RNA libraries were prepared for sequencing using standard Illumina protocols. NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England BioLabs) was used per the manufacturer’s instructions. In general, 1 mg total RNA was subjected to 3’ and 5’ adapter ligation, reverse transcribed, PCR amplified, followed by size selection with AMPure XP beads (Beckman Coulter) for deep sequencing .
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| Library strategy |
ssRNA-seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Raw sequence reads were trimmed with cutadapt 1.18 to remove any adapters while performing light quality trimming with parameters "-a ATCGGAAGAGC -A ATCGGAAGAGC -q 20 --minimum-length=25."
Sequencing library quality was assessed with fastqc v0.11.8 with default parameters and trimmed reads were mapped to the Mus musculus mm10 reference genome using hisat2 2.1.0 with default parameters. Multimapping reads were filtered using SAMtools 1.9. Uniquely aligned reads were then mapped to gene features using subread featureCounts v1.6.2 as a second strand library with parameters. "-t gene -g gene_id -f -p -B -P -C." Differential expression between groups of samples was tested using R version 3.5.1 (2018-07-02) with DESeq2 1.20.0. Transcript quantification was performed with salmon 0.11.3 with parameters "--gcBias --libType A --seqBias --threads 1."
piRNA annotations were derived from the Zamore lab. Transposon-mapping reads were aligned to repBase annotated regions, upbuilt from mm9 to mm10, using the software pipeline piPipes and bowtie2 2.2.5.
s1.txt and s2.txt files are paired-end sequencing results
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimited text files include TPM values for each sample.
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| Submission date |
Aug 29, 2019 |
| Last update date |
Aug 31, 2019 |
| Contact name |
Heejin Choi |
| E-mail(s) |
choih5@nih.gov
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| Phone |
3015941405
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| Organization name |
NIH
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| Street address |
50 South drive, Building 50, Room 3131
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| City |
Bethesda |
| State/province |
Maryland |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE136603 |
Severe sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA |
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| Relations |
| BioSample |
SAMN12655517 |
| SRA |
SRX6773236 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4052571_Homo-1.cutadapt.bam.featurecounts.s1.txt.gz |
849.0 Kb |
(ftp)(http) |
TXT |
| GSM4052571_Homo-1.cutadapt.bam.featurecounts.s2.txt.gz |
869.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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