|
| Status |
Public on Jul 23, 2021 |
| Title |
wild type (cwo5073 control) ZT6 rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Heads
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: wild-type
strain: w1118 strain
|
| Treatment protocol |
Flies were entrained under a condition of 12 hr light/12 hr dark (LD) cycles at 25oC for 3 days prior the collection at ZT2 and ZT14 on the fourth day.
|
| Growth protocol |
Flies were reared on standard cornmeal/agar medium supplemented with yeast and kept in 12 hr light/12 hr dark (LD) cycles at 25oC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using Trizol (Invitrogen), treated with a TURBO™ DNase (Thermo Fisher Scientific), precipitated and purified with Lithium Chloride (Thermo Fisher Scientific) following the manufacture’s instructions.
1.0 μg of total RNA was used to isolate mRNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs), which was used for RNA library construction with the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs). The resulting cDNA products were then used as template for PCR amplification for 12 cycles following the PCR conditions in the manufacturer’s instructions, and after purification the eluted DNA fragments were sent for sequencing. RNA (cDNA) libraries were mixed and multiplexed at the same equimolar concentrations and sequenced using Illumina Next Seq 500 system using 75bp single end reads
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Basecalls performed using RTA v2 software
Reads were mapped to the Drosophila melanogaster genome (dm6 – UCSC) with STAR aligner version 2.6.1d using the following parameters STAR --runThreadN 16
mapped reads were assembled and expression levels were quantified using Strigtie v1.3.5 following parameters described in Pertea et al., Nature Protocols. 2016. Ballgown input table files were generated in StringTie using -B option
A Python script (prepDE.py) was used to extract the read count information from the GTF files generated previously with StringTie. This matrix with read count values for all genes was used to estimate differential expression across all time points using DesSeq2
In parallel, to estimate rhythmicity of genes, the Ballgown object was filtered to remove low expressed genes (using a variance filter to remove transcripts with a variance across all samples less than one, as described in Pertea et al 2016) and as a result a matrix of 8489 genes was created with normalized FPKM values using the function gexp.
This matrix with FKPM was used to estimate significant rhythms using the programs RAIN and MetaCycle and and only genes with an adjusted p-value ≤ 0.05 and fold-change ≥ 1.3 were considered rhythmic.
Genome_build: Drosophila melanogaster genome (dm6 – UCSC)
Supplementary_files_format_and_content: Matrix table with read count values for genes combined for all samples that was used to estimate differential expression with DESeq2.
Supplementary_files_format_and_content: Matrix table with gene-level expression values (FPKM) combined for all samples that was used to evaluate rhythmic expression with RAIN and MetaCycle.
Supplementary_files_format_and_content: Ballgown input tables (from t_data CTAB file but saved here as a txt file) for each sample and replicate, with transcript-level expression values (FPKM).
|
| |
|
| Submission date |
Jan 18, 2021 |
| Last update date |
Jul 24, 2021 |
| Contact name |
Paul Hardin |
| E-mail(s) |
[email protected]
|
| Phone |
4075419203
|
| Organization name |
Texas A&M University
|
| Street address |
3258 TAMU Dept of Biology Buttler Hall room 100H
|
| City |
College Station |
| State/province |
Texas |
| ZIP/Postal code |
77843 |
| Country |
USA |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE165043 |
CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the Drosophila ortholog of CLOCK INTERACING PROTEIN, CIRCADIAN (RNA-Seq) |
| GSE165044 |
CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN |
|
| Relations |
| BioSample |
SAMN17375673 |
| SRA |
SRX9891445 |
