GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM5958713

Query DataSets for GSM5958713

Status

Public on Jan 27, 2023

Title

V_6_A

Sample type

SRA

Source name

head

Organism

Drosophila melanogaster

Characteristics

timepoint: 6
tissue: head
treatment: virgin
genotype: Canton-S

Treatment protocol

Females were mated to a wildtype of Sex Peptide null male within a 3 hour window in the morning (8 AM - 11 AM) and were frozen at their designated time point after mating. Virgin females were frozen in parallel.

Growth protocol

Flies were maintained on yeast/glucose food at 25⁰C on a 12h light/dark cycle (lights on at 8 AM)

Extracted molecule

polyA RNA

Extraction protocol

Flash frozen flies were vortexed to dislodge heads. Heads were collected in Trizol. RNA was extracted and DNA was removed using the Directzol RNA microprep kit (Zymo).
Poly(A)-selected, stranded libraries were made using the NEBnext Ultra II Directional RNA library prep kit and the Poly(A) mRNA magnetic isolation module (New England Biolabs, MA).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Read trimming: Sequencing adaptors were removed using Trimmomatic (version 0.39), which was also used to trim low quality bases at the 5’ end of reads (using parameters TRAILING:20 and SLIDINGWINDOW:5:20). Reads with a minimum length of 30 bases were retained.
Alignment: STAR (version 2.7.5a)
Read counting on gene-level: HTSeq (version 0.11.2); Read counting on exon level: scripts provided as part of R package DEXSeq (Anders et al. 2012).
Each sample was sequenced five times. We used PCA to verify the absence of a sequencing lane effect, after which we merged all five fastq files of each sample and repeated alignment and read counting based on the merged fastq files.
Gene-level filtering: Raw counts were filtered to keep only genes with at least three counts per million in at least three samples and genes encoded in mitochondrial DNA were removed. We removed three samples that were outliers in PCA (C_V_6, C_SP+_2 and C_SP+_3). Exon-level filtering: As a first filtering step, we only kept exons from genes that were analyzed in the gene-level analysis and removed exons that could not be unambiguously assigned to only one gene. For the second filtering step, after normalization (see below), we removed 20% of exons with the lowest normalized counts. Finally, we removed exons that are shared across all transcript isoforms of their gene, or that are present in a gene that has only one annotated transcript isoform. As for the gene-level analysis, we removed three outlier samples (C_V_6, C_SP+_2 and C_SP+_3).
Gene-level normalization: counts per million were calculated in edgeR, using TMM normalization. Exon-level normalization: exons were normalized as recommended in Soneson et al. 2016.
Batch effect correction: to correct for high immune gene expression in a random subset of samples, Combat-seq was applied to both gene and exon counts.
Assembly: dm6 version r6.24 for gene-level alignment; BDGP6.32 for exon-level alignment
Supplementary files format and content: Matrix table with raw gene-level and exon-level read counts. Both gene and exon read counts were corrected for batch effects using ComBat-seq.

Submission date

Mar 17, 2022

Last update date

Jan 27, 2023

Contact name

Sofie Delbare

Organization name

NYU Langone

Street address

435 East 30th Street

City

New York