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Sample GSM1517420 Query DataSets for GSM1517420
Status Public on Oct 01, 2014
Title dme_Embryo_2-3h_spike-in
Sample type SRA
 
Source name embryo
Organism Drosophila melanogaster
Characteristics genotype/variation: w1118
developmental stage: embryo 2-3h
Extracted molecule total RNA
Extraction protocol Small RNA cDNA libraries were generated using TruSeq small RNA sample preparation kit (Illumina) according to manufacturer’s guide. Four synthetic RNA oligos were used for spike-ins, after 5’end phosphorylation by T4 PNK. Four different amount of spike-ins (0.5, 2,4,8 fmole) were added to 10ug total RNA of each sample. For each library, 10 ug of total RNA was separated on 15% urea-PAGE. RNA of 17-29 nt was gel-purified and then ligated to the 3’ adaptor with T4 RNA ligase2 truncated (NEB). After gel purification of 3’ adaptor-ligated RNA, the 5’ adaptor ligation was performed using T4 RNA ligase1. The ligation product was reverse-transcribed by SuperScript II (Life Technologies) and amplified by PCR with Phusion DNA polymerase. The cDNA libraries were sequenced by MiSeq.
Total RNA was extracted using TRIzol reagent for each experimental condition.
 
Library strategy miRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina MiSeq
 
Description small RNA-Seq
Data processing The base calling was done by Illumina Pipeline (CASAVA) v1.8.2.
The 3′ adaptor sequences starting with “TGGAATTC” were removed, and the sequence reads with short length (<16 nt) or artifacts (e.g. long homopolymer) or low quality (Phred quality <30 in >15% of nucleotides) were filtered out using the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/).
Filtered sequence reads from Drosophila melanogaster and Danio rerio were aligned to dm3 and danRer7 reference genomes from UCSC, respectively. The BWA short-read aligner (Li and Durbin, 2009) version 0.7.5a-r405 was used for the alignment, with options of ‘18nt long seed length’ and ‘no allowed mismatches in the seed region.
Each aligned read was classified using intersectBed in Bedtools (Quinlan and Hall, 2010) with annotations retrieved from RefSeq, GtRNAdb, FlyBase, RepeatMasker, Rfam, and miRBase.
We collected reads that perfectly match to the mature miRNA sequences annotated in miRBase as well as those that have 3’ additions to the perfectly matching sequences. Sample-wise normalization of miRNA read counts was done by TMM normalization (Robinson and Oshlack, 2010) using edgR package (http://www.bioconductor.org/packages/release/bioc/html/edgeR.html).
Genome_build: dm3 (GCF_000001215.2); danRer7 (GCA_000002035.2)
Supplementary_files_format_and_content: *.classcount.csv : comma separated files containing sequencing statistics. Column class indicates each class of RNA. Column reads indicates number of sequence reads mapped on each class of RNA. Column proportion indicates the read count proportion to the total pre-processed reads of each class of RNA; dme_miRNA-TMM.csv : comma separated files containing expression level of miRNAs normalized by TMM. Column hairpin indicates name of each mature miRNA. Rest of the columns indicates the normalized expression levels of each mature miRNA in each sample.
 
Submission date Sep 30, 2014
Last update date May 15, 2019
Contact name Yeon Choi
E-mail(s) kitechoi@snu.ac.kr
Phone +82-2-887-1343
Organization name Seoul National University
Department School of Biological Sciences
Lab Narry Kim Lab
Street address Building 504 Room 501, School of Biological Sciences, Seoul National University, 1 Gwanangno, Gwanak-gu
City Seoul
ZIP/Postal code 151-742
Country South Korea
 
Platform ID GPL16479
Series (1)
GSE61931 Adenylation of maternally inherited microRNAs by Wispy
Relations
BioSample SAMN03085466
SRA SRX718005

Supplementary file Size Download File type/resource
GSM1517420_dme_Embryo_2-3h.classcount.csv.gz 546 b (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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