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Sample GSM2537283

Query DataSets for GSM2537283

Status

Public on Jun 04, 2018

Title

Pcl_Su(z)12_RNASeq_rep3

Sample type

SRA

Source name

fly muscle

Organism

Drosophila melanogaster

Characteristics

tissue: muscle
age: 30 day
genotype: Pcl421; Su(z)12253

Treatment protocol

Fly muscles were harvested at the given age.

Growth protocol

Flies were cultured in standard media at 25ºC with 60% humidity unless otherwise specified. The control (WT) line used was 5905 (FlyBase ID FBst0005905, w1118). The given genotype male flies were collected at the day of eclosion and transferred to new vials every other day during aging process.

Extracted molecule

polyA RNA

Extraction protocol

For ChIP-Seq, 5% mouse epigenome was used as internal control for H3K27me3 IP.
For RNA-Seq, RNA was isolated by TRIZol reagent, and polyA tail RNAs were selected.
For small RNA-seq, ~20-29 nt small RNA was recoveried by 15% TBE-UREA PAGE gels.
ChIP libraries were prepared according to instructions of NEB DNA library prep kit (NEB, E7370L). RNA libraries were prepared according to instructions of NEB Ultra RNA library prep kit (NEB, E7530L). Small RNA libraries were prepared by NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S).
ChIP-Seq or RNA-Seq or miRNA-seq

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 550

Description

WT_vs_Pcl_Su(z)12_DESeq.csv

Data processing

For ChIP-Seq, Sequencing reads were mapped to reference genome dm6 or mm10 respectively with Bowtie2 by default parameter. The peak regions were identified by homer function findPeaks with parameter –style histone -F 2 -size 3000 -minDist 5000. The overlapped peaks of replicate data-sets were found using homer function mergePeaks by default parameter. Bedtools were used to compare the peaks between different genotypes and aging stages. To get inter-peak regions, we used Bedtools to subtracted the peak regions from whole genome.
For quantitative comparison, the dm6 mapped reads were normalized to a scale factor using deeptools function bamCoverage with 10 bp bin size. The scale factor for each sample was calculated as reported (David A. Orlando et al., 2014) with a small modification. The percentage of mouse genome in input was set to the mm10 mapped reads in total mapping reads, rather than a constant. The ChIP intensity of each genes or regions were calculated using the Bwtool function bwtool summary.
For RNA-Seq comparison, Sequencing reads were mapped to dm6 with STAR by default parameter. The read counts for each genes were calculated by HTSeq-count. The count files were used as input to DESeq for differential expression analysis.
For small RNA-Seq, adapters were removed by fastx, microRNAs were identified by miRDeep2.
Genome_build: dm6 and mm10
Supplementary_files_format_and_content: bigwig files were generated by deeptools. DESeq was used for calling differential expression.

Submission date

Mar 15, 2017

Last update date

May 15, 2019

Contact name

Hui Wang

E-mail(s)

wanghuisimm@sioc.ac.cn, mazj@sioc.ac.cn

Organization name

Chinese Academy of Science

Street address

Qiuyue Road 26