 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 18, 2017 |
| Title |
kmg KD testis rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Testis
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: UAS-RNAi-kmg (VDRC# 107395); bam-gal4, UAS-dicer2
age: 0~1 day post eclosion
|
| Growth protocol |
Crosses and experimental flies were grown on cornmeal and molasses media at 30 °C
|
| Extracted molecule |
total RNA |
| Extraction protocol |
kmg ctrl, kmg KD, dMi-2 ctrl, and dMi-2 KD testes without accessory glands were dissected in PBS for 30 minutes and immediately frozen in liquid nitrogen before transferred to -80°C. Wild-type heads were manually decapitated from the body and frozen and stored as in testes preparation. Total RNA was extracted using TRIzol (Invitrogen). RNA was further purified by RNeasy mini column (Qiagen) to minimize residual salts. RNA quality was assessed by checking rRNA integrity using Bioanalyzer. ~30ug of total RNA was used to purify polyA RNA by using Poly(A)Purist MAG Kit (Thermo Fisher, Cat#AM1922).
100ng of purified mRNA was used to make a strand-specific sequencing library by dUTP incorporation method using KAPA Stranded RNA-Seq Library Preparation Kit Illumina® Platforms (KAPA Biosystems, Cat#: KK8400) following manufacturer’s instructions.
All RNA-Seq libraries were sequenced by NextSeq Mid Output mode 150bp paired end. Between 3 to 4 samples were pooled in a single lane, yielding on average 48 million reads per sample (34 to 65 million). All sequencing was performed by the Stanford Functional Genomics Facility (SFGF).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
polyA RNA purified from total RNA by oligodT beads
|
| Data processing |
fastq files were generated using bcl2fastq (v2.17.1.14) from Illumina
Trimming: all raw sequencing data were quality and adapter trimmed by using a wrapper script TrimGalore that combines Cutadapt (ver. 1.8.1) and FastQC (ver. 0.11.4) with adapter overlap stringency of 1 and Phred quality score cutoff of 20.
Mapping: trimmed fastq files were mapped using a splice-aware aligner, Tophat (ver. 2.0.9), utilizing Bowtie2 (ver. 2.1.0) to the Drosophila melanogaster genome (release 6.07) while supplying trancript annotation obtained from the Ensembl database (Drosophila_melanogaster.BDGP6.82.chr.gff3). Default parameters were used, except the library type was specified as ‘fr-firststrand’ since only the first strand was retained by dUTP method in library preparation.
Post mapping: the bam files generated by mapping were further filtered to exclude unmapped reads (-F 0x04), and reads mapped with low confidence (-q 1) using SAMtools (ver. 1.2) (48). Filtered bam files were sorted and indexed by using SAMtools.
Read counts calculation and visualization: to obtain read counts corresponding to genomic positions, bedgraph files were generated from bam files using the BEDTools genomecov (50) while normalizing all the samples to have the equivalent of 1 million mapped reads using '-scale' and '-split' options. Given the sequencing library is made with dUTP method, Bam files were divided and re-merged to obtain strand specific read counts. Bedgraph files were converted into bigwig files using the bedGraphToBigWig program.
Genome_build: dm6 (release 6.07 downloaded from the Flybase)
Supplementary_files_format_and_content: Bigwig files which can visualize strand specific read abundances in genome browsers
|
| |
|
| Submission date |
Nov 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert E Kingston |
| E-mail(s) |
kingston@molbio.mgh.harvard.edu
|
| Organization name |
Massachusetts General Hospital
|
| Department |
Molecular Biology
|
| Lab |
Kingston lab.
|
| Street address |
185 Cambridge Street
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE89380 |
Gene expression of fly testes with dMi-2, kumgang (CG5204) knock downs and wild-type heads by RNA-Seq |
| GSE89506 |
Blocking promiscuous activation at cryptic promoters directs cell type–specific gene expression |
|
| Relations |
| Reanalyzed by |
GSM3282211 |
| BioSample |
SAMN05978655 |
| SRA |
SRX2325626 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2367701_JK2523_kmg-KD-02-CGATGT_S2_forward.bw |
72.6 Mb |
(ftp)(http) |
BW |
| GSM2367701_JK2523_kmg-KD-02-CGATGT_S2_reverse.bw |
73.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
