|
| Status |
Public on Apr 13, 2020 |
| Title |
Yellow-white_WingDisc_RNA_resequence_Replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
female 3rd instar larval wing disc
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype/variation: Yellow-white
tissue: Wing disc
Sex: Female
developmental stage: 3rd instar larvae
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from 40 3rd instar wing imaginal discs. Wing imaginal discs were homogenized in Trizol (Invitrogen) and flash frozen in liquid nitrogen. RNA was isolated using the Direct-zol RNA miniprep kit (Zymo Research).
rRNA was depleted using the RiboMinus™ Eukaryote Kit for RNA-Seq (Invitrogen) and libraries were prepared using the NEBNext® Ultra™ II RNA Library Prep Kit.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Reads from G1 and S samples were aligned to the dm6 reference genome (Release 6.04) using Bowtie 2 (v2.3.2) default parameters (Langmead et al. 2009).
Reads with a MAPQ score greater than 10 were retained using SAMtools (v1.9) (Li et al. 2009).
txtTools coverage (v2.26.0) was used to quantify the number of reads mapping to each 100kb window, with results normalized to read depth (Quinlan and Hall 2010).
Replication timing (RT) values were obtained by averaging the S/G1 ratio of reads per million (RPM) value from each S phase replicate for a particular window size. Profiles were generated by plotting the RT value at each window versus genomic location.
Quantile normalization was performed for comparisons between samples through the preprocess Core R package to equalize the dynamic range of RT values (Bolstad 2016).
The limma statistical package was used to identify 100kb windows with significantly altered RT values (lmFit, p value adjusted for multiple testing (p<0.01); absolute log2 fold change > 0.1) (Newville et al. 2014).
BEDTools intersect (v2.26.0) was used to determine overlap of 100kb windows with -f 0.5 and -u parameters (Quinlan and Hall 2010).
TopHat default parameters (v2.1.1) (Trapnell et al. 2012) were used to align single-end reads to the dm6 version of the Drosophila genome.
Transcriptomes were generated using Cufflinks (v2.2.1, see supplementary materials for parameters). We combined the Cufflinks generated transcriptome with transposons annotated by RepeatMasker (Smit et al. 2013-2015).
Differentially expressed transcripts were determined via edgeR statistical analysis (p value <0.01) (Robinson et al. 2010; McCarthy et al. 2012).
We assigned the RT log2 fold-change and associated limma-generated adjusted p-value determined at 10kb windows to individual transcripts; if a transcript overlapped multiple 10kb windows, we assigned the average RT log2 fold change and average adjusted p-value of all from all overlapping windows to that transcript.
Genome_build: dm6 (Release 6 plus ISO1 MT)
Supplementary_files_format_and_content: all_raw_avgRT_values.txt, bigWig files
|
| |
|
| Submission date |
Dec 08, 2019 |
| Last update date |
Apr 13, 2020 |
| Contact name |
Jared Nordman |
| E-mail(s) |
jared.nordman@vanderbilt.edu
|
| Organization name |
Vanderbilt University
|
| Street address |
465 21st Ave South MRB III 4268
|
| City |
Nashville |
| State/province |
Tennessee |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platform ID |
GPL23702 |
| Series (1) |
| GSE141632 |
Rif1 functions in a tissue-specific manner to control replication timing through its PP1-binding motif |
|
| Relations |
| BioSample |
SAMN13508673 |
| SRA |
SRX7286051 |
