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Sample GSM6777762 Query DataSets for GSM6777762
Status Public on Oct 23, 2023
Title N2_WT_RNAseq
Sample type SRA
 
Source name Testes
Organism Mus musculus
Characteristics immunoprecipitation: n/a
genotype: WT
mouse: Mouse-17079
Growth protocol The reporters were created by inserting one or ten complementary binding sites for piR-A in the 3ʹ UTR of the Ythdc2 gene (MGI: 2448561; NCBI Gene: 240255). Binding sites were perfectly complementary or contained two mismatches at the 10th and 11th position relative to the guide RNA. Mismatched sites are referred to bulges. The first line contains one perfect complementary binding site (1XPerf), the second line contains one bulge binding site (1xBulge), the third line contains ten perfect complementary binding sites (10xPerf) and the fourth line contains ten bulge binding sites (10xBulge). Ythdc2 locus of the B6D2F1/J hybrid line (also called B6D2; The Jackson Laboratory, stock no. 100006) was targeted using CRISPR/Cas9 system.
Extracted molecule total RNA
Extraction protocol Total RNA was extracted from adult testes using TRIzol™ Reagent (Thermo Scientific, Cat. No. 15596026). Immunoprecipitation was conducted with Mili (PMID: 19465913) or Miwi antibodies (PMID: 22121019) and Protein G Sepharose® 4 Fast Flow beads (GE Healthcare, No. 17-0618-01) and Mili and Miwi loaded piRNAs were extracted using phenol-chloroform.
piRNA sequencing libraries were prepared with NEBNext® Multiplex Small RNA Library Prep Set for Illumina® (New England Biolabs, Ref. E7300L) and sequenced by NextSeq 500 platform (Illumina) in GeneCore(Heidelberg). The Stranded Total RNA Ribo-Zero Plus kit from Illumina was used for the library preparation from total RNA with 500 ng of total RNA as input. Library molarity and quality were assessed with the Qubit and Tapestation (DNA High sensitivity chip). Libraries were sequenced on a NovaSeq 6000 Illumina sequencer for SR100 reads (iGE3 Genomics Platform, University of Geneva).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
 
Description all_genes.csv.gz
Data processing Reads from small RNA libraries were sorted into individual libraries based on the barcodes and the 3′ adapter sequences were clipped from the reads using cutadapt (DOI:http://dx.doi.org/10.14806/ej.17.1.200) with only sequences of at least 15 nucleotides left for further analysis (cutadapt parameters: -a AGATCGGAAGAGCACACGTCT -m 15 -e 0.2 -O 4 -q 10 --match-read-wildcards). The reads were then mapped to the mouse genome (GRCm38: Ensembl release 95) using STAR(Dobin et al., 2013) (with parameters: --runThreadN 10 -outFilterType BySJout --limitOutSJcollapsed 50000000 --limitIObufferSize 1500000000) and length distributions of the reads were plotted. Read counts were normalized to library sizes. To see whether any piRNAs are produced from the reporters, the reads were mapped by bowtie(Langmead et al., 2009) (with parameters: -v 0 -a --best --strata) to the Ythdc2 transcript sequence containing individual reporters: 1xPerf,1xBulge,10xPerf,10xBulge.
Reads from long RNA-seq libraries were mapped to the mouse genome (GRCm39 - Ensembl release 104) using salmon(Patro et al., 2017) (salmon quant with options -l A –validateMappings–gcBias). Further analysis was performed using R(R Core Team, 2017) and Bioconductor(Huber et al., 2015). The DESeq2(Love et al., 2014) package was used to obtain normalized read counts for individual genes.
Assembly: GRCm38(small RNA libraries) and GRCm39 (total RNA libraries)
Supplementary files format and content: The bigwig files contain piRNA coverages along the genome. The csv file contains gene expression data.
 
Submission date Dec 01, 2022
Last update date Oct 23, 2023
Contact name Ramesh Pillai
E-mail(s) ramesh.pillai@unige.ch
Organization name University of Geneva
Department Department of Molecular Biology
Street address 30, Quai Ernest-Ansermet
City Gneveva
ZIP/Postal code CH-1211
Country Switzerland
 
Platform ID GPL24247
Series (1)
GSE219200 In vivo PIWI slicing in mouse testes deviates from rules established in vitro
Relations
BioSample SAMN31977761
SRA SRX18456161

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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