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| Status |
Public on Oct 31, 2017 |
| Title |
NIPP1_KO_testis_mouse4 |
| Sample type |
SRA |
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| Source name |
NIPP1 KO_6 wks_tamoxifen treated_testis
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| Organism |
Mus musculus |
| Characteristics |
strain background: mixed
genotype/variation: NIPP1 KO; UBC CRE-ERT2+/- Ppp1r8 fl/-
age: 6 weeks
treated with: tamoxifen
tissue: total testis
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| Treatment protocol |
Treatment with tamoxifen (0.2 mg/g mouse; 4 intraperitoneal injections with the first injection at the age of 4 week) prior to testis removal and RNA extraction
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| Growth protocol |
Mice housed in a SPF facility; 12h light/dark cycles; water and food at libitum
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| Extracted molecule |
total RNA |
| Extraction protocol |
Adult testes were harvested 2 weeks after treatment with tamoxifen. RNA extracted from 40 mg of testis; GenEluteTM Mammalian Total RNA Miniprep kit (SIGMA). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent).
Per sample, an amount of 5 µg of total RNA was used as input. Using the Illumina TruSeq® Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (protocol version Rev E. “October 2013”) rRNA is depleted from the total RNA samples using Ribo-Zero ribosomal RNA reduction chemistry. Subsequently, RNA was purified and fragmented and converted into first strand cDNA in a reverse transcription reaction using random primers. Next, double-stranded cDNA was generated in a second strand cDNA synthesis reaction using DNA Polymerase I and RNAse H. The cDNA fragments were extended with a single ‘A’ base to the 3’ ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally an enrichmend PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence-libraries of each sample were equimolarly pooled and sequenced on ½ run of a NextSeq500 flow-cell at 2x75 bp.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
TKO4
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| Data processing |
Read trimming and filtering: Low quality ends and adapter sequences were trimmed off from the Illumina reads with FastX 0.0.13 and Cutadapt 1.7.1. Using FastX 0.0.13 and ShortRead 1.16.3, we filtered subsequently small reads (length < 35 bp), polyA-reads (more than 90% of the bases equal A), ambiguous reads (containing N) and low quality reads (more than 50% of the bases < Q25). With Bowtie2 v2.2.4 we identified and removed reads that mapped to the spiked-in PhiX. Only fragments with both a read1 and read2 were kept. The number of processed fragments (i.e. paired reads) per sample then varied between 21,692,611 and 24,834,067.
Mapping: Processed reads were aligned with Tophat v2.0.13 to the reference genome of Mus musculus (GRCm38.73), as downloaded from the Genome Reference Consortium. Default Tophat parameter settings were used, except for ‘library-type=fr-firststrand’, ‘mate-inner-dist=50’, ‘mate-std-dev=20’, 'min-intron-length=50', 'max-intron-length=500,000', 'no-coverage-search', ‘no-mixed’ and 'read-realign-edit-dist=3'. Using Samtools 1.1, reads with a mapping quality smaller than 20 were removed from the alignments.
Counting: Transcript coordinates were extracted from the GRC reference annotation with Gffread from the Cufflinks v2.1.1 suite and merged to gene coordinates with mergeBed from the Bedtools v2.17.0 toolkit. The numbers of aligned reads per gene were summarized using HTSeq-count v0.6.1p1 with parameters 'm=union', 'stranded=reverse', 'a=0', 't=exon' and 'i=gene_id'.
Genome_build: GRCm38
Supplementary_files_format_and_content: One tab-delimited text file with raw read counts per gene and per sample. Gene IDs are Ensembl IDs.
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| Submission date |
Jun 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
Nucleomics.Bioinformatics@vib.be
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| Organization name |
VIB
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| Department |
Nucleomics Core
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| Street address |
Herestraat 49 Box 816
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| City |
Leuven |
| ZIP/Postal code |
B-3000 |
| Country |
Belgium |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE83145 |
RNA profiling of testis from wild-type and tamoxifen-induced NIPP1 knockout mice |
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| Relations |
| BioSample |
SAMN05220578 |
| SRA |
SRX1835335 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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