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Sample GSM4555333

Query DataSets for GSM4555333

Status

Public on May 16, 2020

Title

nRNAseq_5minLight3

Sample type

SRA

Source name

mushroom body

Organism

Drosophila melanogaster

Characteristics

line: CS10
tissue: mushroom body
treatment: 5-min optogenetic activation

Treatment protocol

Flies were subjected to optogenetic activation by expressing CsChrimson with red-light illumination.

Growth protocol

Flies were raised under a 12 h:12 h, light:dark cycle, at 25℃ and 60% humidity.

Extracted molecule

total RNA

Extraction protocol

Heads were collected and homogenized in crosslinking buffer. The nuclei were dissolved in extraction buffer, sonicated to dissociate the individual nuclei, and precipitated with ANTI-FLAG M2 Affinity Gel. The precipitates were rinsed 4 times in extraction buffer, with 5 min nutation at 4℃ between washes, and subjected to ChIP analysis or RNA extraction for RNA-seq.
The library for ChIP-seq was prepared by NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs inc. Ipswich, MA, USA), and that for nuclear RNA-seq was prepared by Oligo d(T)25 Magnetic Beads (New England Biolabs inc. Ipswich, MA, USA), and NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs inc. Ipswich, MA, USA), according to the manufacturer’s instructions.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

HiSeq X Ten

Data processing

The adaptors were trimmed via Trimommatic, followed by mapping to the Drosophila reference genome, dm6 from UCSC using STAR.
The reads with low mapping quality below 8 and the non-primary mapped reads were eliminated.
MACS was used for peak calling in ChIP-seq on Strand NGS software, using a default setting except for the following parameters; 10-4 as a P-value cutoff, and 3 as a enrichment factor.
The binding sites were determined when the peaks were overlapped in at least 2 out of 3 biological replicates.
For RNA-seq, the filtered reads were analyzed with HTSeq-count to obtain numbers of the reads mapped on exons, which was further analyzed on R using DESeq2
Genome_build: dm6
Supplementary_files_format_and_content: peak text files
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every sample
Supplementary_files_format_and_content: tab-delimited text files include statistical results and fold enrichment comparing to naive samples

Submission date

May 15, 2020

Last update date

May 18, 2020

Contact name

Yukinori Hirano

E-mail(s)

yukinori@ust.hk

Organization name

The Hong Kong University of Science and Technology

Department

LIFS