GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM6034547

Query DataSets for GSM6034547

Status

Public on Mar 06, 2024

Title

small RNA human 2

Sample type

SRA

Source name

oocyte

Organism

Homo sapiens

Characteristics

tissue: oocyte
Sex: female
Stage: GV
genotype: wild type
molecule subtype: small RNA

Extracted molecule

total RNA

Extraction protocol

Total RNA from oocytes of different species were extracted with TRIzol Reagent (Ambion, USA), respectively.
Small RNA library construction. Briefly, the single oocytes were incubated at 72 °C for 3 min then cooled in ice. After 3′ adapter ligation, the samples were incubated with 5 U of lambda exonuclease and 25 U of 5′ de-adenylates. Small RNAs were reverse-transcribed after 5′ adapter ligation. Two rounds of amplification were conducted to get the libraries and the libraries were recovered with 6% polyacrylamide gel.
mRNA library construction. In brief, single oocytes were incubated at 72 °C for 3 min and then cooled on ice. For every single oocyte, 1 μl of a 1/500,000-1/50,000 dilution of the ERCC RNA Spike-In Mix (Invitrogen, 4456740) was added. After reverse transcription and PCR pre-amplification, cDNAs were purified and 3 ng of purified cDNA was used for a tagmentation reaction with Tn5 transposase.

Library strategy

miRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina NovaSeq 6000

Data processing

We used cutadapt to clip adaptor and filter low quality reads. Reads failing to match the adaptor or reads with lengths shorter than 17 nt were discarded. Redundant sequences were collapsed as useful reads and mapped to reference sequences by bowite. The useful reads were assigned to known miRNAs, tsRNA (tRNA-derived small non-coding RNA), rsRNA (rRNA-derived small non-coding RNA), small snoRNAs, lncRNA, and mRNA, successively. The reads that could not be mapped to these known small RNAs were used to predict piRNAs and endo-siRNAs successively. Sequences that were not annotated with any of the RNA categories above were classified as others.
We used trimmomatic to clip adaptor and filter low quality reads. The high quality reads were mapped to reference genomes by STAR. The expressed levels of genes were caculated in TPM (transcripts per kilobase of exon model per million mapped reads) by StringTie.
Assembly: GRCh38, GRCm38
Supplementary files format and content: tab-delimited text files include TPM values or raw counts for each Sample

Submission date

Apr 08, 2022

Last update date

Mar 06, 2024

Contact name

Wei Liu

E-mail(s)

liuwei2018@sibcb.ac.cn

Organization name

Chinese Academy of Sciences

Department

Institute of Biochemistry and Cell Biology