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Sample GSM5530729

Query DataSets for GSM5530729

Status

Public on Sep 27, 2022

Title

smallRNA-seq CTRL Spc 3

Sample type

SRA

Source name

testis

Organism

Mus musculus

Characteristics

cell type: isolated pachytene spermatocytes
genotype: control
strain: mixed C57Bl/6J and SV129

Extracted molecule

total RNA

Extraction protocol

RNA was extracted from enriched fractions of Spc, RS or isolated CBs with the Trisure reagent (Bioline) using standard protocols. Isolated RNA was analyzed using NanoDrop (Thermo Scientific) and Bioanalyzer (Agilent).
1) CB RNA-seq: Total RNA libraries without ribosomal RNA removal were prepared by NEBNext® Ultra™ Directional RNA Library Prep Kit without fragmentation. Sequencing was performed with NovaSeq6000 (PE150) platform (Illlumina) at Novogene (www.en.novogene.com). 2) RNA-seq of Spc and RS samples: Libraries were constructed with Illumina TruSeq Stranded mRNA library preparation kit and sequenced with HiSeq3000 (read length 2 x 75 bp) sequencing system (Illumina, San Diego, CA, USA) at the Finnish Functional Genomics Centre (https://bioscience.fi/services/functional-genomics/services/). 3) SmallRNA-seq of Spc and RS samples: Libraries were constructed with QIAseq miRNA library preparation kit and sequenced with HiSeq2500 Rapid run sequencing platform (1 x 75bp) at the Finnish Functional Genomics Centre.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 3000

Description

counts_piRNA_cluster_smallRNAseq.txt
counts_piRNA_rmsk_smallRNAseq.txt
merge_miRNAs_smallRNAseq.txt

Data processing

RNA-seq: For transcript-level DE analysis, reads were trimmed off adapter contents using cutadapt (v2.8) and mapped to the mouse genome (Ensembl: Grcm38) using STAR (v2.7.1a) in two-pass mode. Then, reads were assigned and counted using FeatureCounts (v2.0.1) in paired-end mode. First, raw read counts were filtered in order to keep only transcripts with 20 or more normalized counts in at least three individual samples among six samples from a given cell type (3 x CTRL and 3 x cKO). Raw count reads were normalized and differential expression calculated using DESeq2 in R (v4.0.3). 2) To analyze transposable elements and piRNA precursors, reads were mapped to the mouse genome (UCSC: mm10) using STAR (v2.7.1a) in two-pass mode. For transposable elements, reads were assigned and counted using TEtranscripts in reverse stranded mode (v2.2.1). For piRNA precursor transcripts, reads were assigned and counted against the gtf file containing the 214 piRNA precursors using FeatureCounts. Raw count reads were filtered to keep only genes with at least 10 reads across all samples (Smg6-cKO and CTRL). Raw count reads were normalized and differential expression calculated using DESeq2 in R (v4.0.3).
Small RNA-seq: SPORTS1.1 was used to map small RNA reads successively rsRNA, miRNA, tsRNA and piRNA sequences extracted from mm10 UCSC genome files originating from rRNAdb, miRBase (v.22) GtRNAdb (v.2.0) and piRBase (v.2.0) using SPORTS default settings. Then, reads mapping to rsRNA, miRNA and tsRNA sequences were extracted from SPORTS output text file using R. Repeats from repeatMasker and piRNA precursor locations were mapped to the mouse genome (UCSC : mm10) using HISAT2 (v2.1.0); then, reads were assigned and counted using FeatureCounts (v2.0.1). Raw read counts were filtered to keep only genes with at least one occurrence found per sample. Raw read counts were normalized and differential expression calculated using DESeq2 (v4.0.3).
Genome_build: mm10
Supplementary_files_format_and_content: Raw read counts text files

Submission date

Aug 20, 2021

Last update date

Sep 27, 2022

Contact name

Matthieu Bourgery

E-mail(s)

[email protected]

Organization name

University of Turku

Lab

Noora Kotaja's lab