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Sample GSM4475723

Query DataSets for GSM4475723

Status

Public on Jun 22, 2022

Title

MMB21

Sample type

SRA

Source name

Brain

Organism

Drosophila melanogaster

Characteristics

Sex: Male
cell type: single octopaminergic/tyraminergic neuron, expressing a membrane-bound GFP reporter.
genotype: Mutant
collection strategy: Fluorescent-Activated Cell Sorting

Treatment protocol

Wild-type and nervy-mutant frui flies were reared in groups for 6 days, anestethized on ice and dissected in Schneider's Drosophila medium

Growth protocol

Fruit flies were used. Animals were reared in groups of 15 individuals, from eclosion to day 6 in vials with standard fly medium.

Extracted molecule

total RNA

Extraction protocol

Single cells were sorted via fluorescent-activated cell sorting. Each cell was sorted in a separate well of a 96 well plate filled with freshly made lysis buffer, prepared according to Takara standard protocol. The contact with the lysis buffer lysed the cells, freeing their content, mRNA included. As soon as collection was completed (15-20 minute/genotype), samples were frozen in dry ice and stored until used at -80°C.
Sequencing libraries were prepared using the Nextera XT kit (Illumina #FC-131-1096) and mixed into 24 pools (12 samples per pool).After purification using the Agencourt AMPure XT beads (Beckman Coulter #A63881), the sample quality was checked with both the Qubit 3 Fluorometer and the High Sensitivity D1000 ScreenTape assay (Agilent Technologies #5067-5584). The libraries were equimolarly pooled, and the final concentration was estimated by qPCR. Sequencing of 75 bp paired-end reads was performed with the Illumina Nextseq 500 sequencer.
Nextera XT

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Sequenced reads were quality-tested using FASTQC.
Reads were aligned to the D. melanogaster genome dm6 (from The FlyBase Consortium/Berkeley Drosophila Genome Project/Celera Genomics) using the alignment algorithm STAR84 (version 2.5.3a). Mapping was carried out using default parameters (up to 10 mismatches per read, and up to 9 multi-mapping locations) and --outFilterIntronMotifs RemoveNoncanonical.
Raw gene expression was quantified using the software HOMER across exons, and the top isoform value was used to represent gene expression.
In brief, cells containing the bottom 10% of cell counts were filtered, TMM normalization and size-factor correction was applied using the edgeR package, and cells falling in the bottom 5% of normalized counts were removed. Then, the bottom 10% of genes with normalized counts > 32 in a cell were removed as non-expressed genes.
Expression values were log2 transformed, and tSNE was performed to generate the plots.
The scrattch.hicat R package was used to perform hierarchical iterative clustering on the normalized expression table.
For differential expression analysis between the wild-type and ∆nvy mutant, genes with expression values of 0 in 75% or more of the cells were filtered out. Genes that met the following criteria were considered as DEGs: (1) p-values by Mann-Whitney U-test lower than 0.05, (2) adjusted p-values by Benjamini-Hochberg FDR test lower than 0.2, and (3) the fold-change greater than 10 (|log2FC| > 3.321928095).
For DEGs that showed behavioral phenotypes in the RNAi experiments, predicted biological processes and human orthologs were taken from the “Gene Ontology” and “Human Orthologs (via DIOPT v7.1)” sections in FlyBase (http://flybase.org/), respectively.
Genome_build: dm6
Supplementary_files_format_and_content: raw count table in tab-delimited text format

Submission date

Apr 14, 2020

Last update date

Jun 22, 2022

Contact name

April Elizabeth Williams

E-mail(s)

[email protected], [email protected]

Phone

7345461645

Organization name

Salk Institute for Biological Studies