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Sample GSM4820461

Query DataSets for GSM4820461

Status

Public on Oct 08, 2020

Title

PS16.04.05

Sample type

SRA

Source name

Mature sperm

Organism

Homo sapiens

Characteristics

subject: 04
collection: 5

Extracted molecule

total RNA

Extraction protocol

Mature sperm were enriched by centrifugation through a 50% PureSperm density gradient (Nidacon). Sperm were lysed and homogenized in TRIzol-LS, supplemented with 0.2 M β-mercaptoethanol. Total RNAwas were isolated using Qiagen’s miRNeasy Mini kit.
small RNA libraries were constructed with 10 ng of small RNA using the TruSeq small RNA Library Prep Kit (Illumina) according to manufacturer's protocol.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

NextSeq 550

Data processing

The small non-coding RNA sequencing (sncRNASeq) analytical pipeline was designed using the snakemake framework and is available via GitHub at https://github.com/acshetty/sncRNA-seq-analysis.
Reference transcriptome sequences in FASTA format were downloaded from public repositories and indexed using the Bowtie short read aligner software. These references included the GRCh38 ncRNA reference from Ensembl, the miRNA reference from miRbase 21, the tRNA reference generated from GRCh37 by GtRNAdb 2.0, and the piRNA reference from piRBase v1.0.
Sequenced reads were trimmed at the 3’ end to remove any trailing adaptor sequence using the Trimmomatic tool. Reads shorter than 15 nucleotides were discarded before downstream analyses.
Trimmed reads for each sample were aligned to each of the different sncRNA class-specific reference transcriptomes using Bowtie. Reads were aligned allowing for 2 mismatches and a seed length of 15 nucleotides. The alignment statistics were summarized for each sample across each of the different references and raw expression values were computed based on the number of reads aligned to each of the sncRNAs specified in their respective reference files.
Expression of sncRNA were adjusted for differences in library sequencing depth to generate counts per million reads (CPM) following TMM normalization using the Bioconductor package ‘edgeR’ (version 3.24.3). sncRNA features were retained for analysis if they were expressed at ≥ 1 CPM in at least 75% of samples.
Genome_build: GRCh38 ncRNA reference from Ensembl, the miRNA reference from miRbase 21, the tRNA reference generated from GRCh37 by GtRNAdb 2.0, and the piRNA reference from piRBase v1.0.
Supplementary_files_format_and_content: Comma separated values file containing normalized log2 counts per million reads for each sample

Submission date

Oct 07, 2020

Last update date

Oct 09, 2020

Contact name

Christopher P Morgan

E-mail(s)

[email protected]

Organization name

University of Maryland School of Medicine

Department

Pharmacology