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| Status |
Public on Feb 11, 2023 |
| Title |
Dsim_testis_RNAseq_replicate1 |
| Sample type |
SRA |
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| Source name |
D. simulans testis
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| Organism |
Drosophila simulans |
| Characteristics |
tissue: testis
genotype: wildtype
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| Growth protocol |
All flies were raised in 25C for testis extraction
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| Extracted molecule |
total RNA |
| Extraction protocol |
For small RNA analysis, we extracted RNA from testes and accessory glands of 7-day-old Dsim w[XD1] strain using Trizol (Invitrogen). 1 μg of total RNA was used to prepare small RNA libraries as described3, with the addition of QIAseq miRNA Library QC Spike-ins for normalization (Qiagen). Adenylation of 3' linker was performed in a 40 µL reaction at 65°C for 1 hr containing 200 pmol 3' linker, 1X 5' DNA adenylation reaction buffer, 100 nM ATP and 200 pmol Mth RNA ligase and the reaction is terminated by heated to 85°C for 5 min. Adenylated 3' linker was then precipitated using ethanol and was used for 3' ligation reaction containing 10% PEG8000, 1X RNA ligase buffer, 20 µM adenylated 3' linker and 100 U T4 RNA Ligase 2 truncated K227Q. The 3' ligation reaction was performed at 4°C overnight and the products were purified using 15% Urea-PAGE gel. The small RNA-3' linker hybrid was then subjected to 5' ligation reaction at 37°C for 4 hr containing 20% PEG8000, 1X RNA ligase buffer, 1 mM ATP, 10 µM RNA oligo, 20 U RNaseOUT and 5 U T4 RNA ligase 1. cDNA synthesis reaction was then proceeded immediately by adding following components to the ligated product: 2 μl 5x RT buffer, 0.75 μl 100 mM DTT, 1 μl 1 μM Illumina RT Primer, and 0.5 μl 10 mM dNTPs. The RT mix was incubated at 65 C for 5 min and cooled to room temperature and transfer on to ice. 0.5 µL of superscript III RT enzyme and 0.5 µL RNase OUT were added to the RT mix and the reaction was carried out at 50°C for 1 hr. cDNA libraries were amplified using 15 cycles of PCR with forward and illumine index reverse primers and the amplified libraries were purified by 8% non-denaturing acrylamide gel. Purified libraries were sequenced on HiSeq2500 using SR50 at the New York Genome Center.
We used Dsim w[XD1], which were used for PacBio genome sequencing (Chakraborty et al. Genome Research 2021). We isolated total RNA from ~ 5-day-old flies and for Dsim samples. We extracted RNA from testes (dissected free of accessory glands) using Trizol (Invitrogen). We made two independent dissections to generate biologically replicate RNA samples, whose quality was assessed by Bioanalyzer. We used the Illumina TruSeq Total RNA library Prep Kit LT to make RNA-seq libraries from 650 ng of total RNA. Manufacturer's protocol was followed except for using 8 cycles of PCR to amplify the final library instead of the recommended 15 cycles, to minimize artifacts caused by PCR amplification. All samples were pooled together, using the barcoded adapters provided by the manufacturer, over 2 flow cells of a HiSeq2500 and sequenced using PE75 at the New York Genome Center.
Single-end sequencing for small RNA sequencing and paired-end sequencing for RNA sequencing data
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Dsim testis RNA replicate 1
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| Data processing |
RNA-seq data: Paired-end RNA-seq reads was mapped to PacBio genome assemblies for Dsim using hisat2 aligner with the following command hisat2 -x indexed_genome_assembly -1 $ read1.fastq.gz -2 read2.fastq.gz -S file.sam. The alignment file in SAM format was then converted to a compressed BAM file using SAMTOOLS1 with the following commands: (1) samtools view -bS file.sam > file.bam (2) samtools sort file.bam > file_sorted.bam (3) samtools index file_sorted.bam. Mapping statistics for the BAM alignment files were obtained using bam_stat.py script from the RSeqQC package2.
small RNA data: sRNA reads were processed as follows: Raw sequence reads were adapter trimmed using Cutadapt software (https://cutadapt.readthedocs.io/en/stable/). After clipping the adapter sequence, we removed the 4bp random-linker sequence inserted at 5’ and 3’ of the sRNA sequence (total 8bp). After filtering <=15 nt reads, we mapped the small RNA data to PacBio genome assemblies using Bowtie (with –v0 –best –strata options.) The resulting small RNA alignments in SAM format were converted to BED for downstream processing using the BEDops software and visualized on IGV.
Assembly: Data was mapped to PacBio genome assembly (Chakraborty et al. Genome Research 2021) for D. simulans.
Supplementary files format and content: The processed data files are in bigWig format for viewing in the IGV browser
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| Submission date |
Feb 11, 2023 |
| Last update date |
Feb 11, 2023 |
| Contact name |
Jeffrey Vedanayagam |
| E-mail(s) |
[email protected]
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| Organization name |
Sloan-Kettering Institute
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| Department |
Department of Developmental Biology
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| Lab |
Eric Lai
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| Street address |
1275 York Avenue
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| City |
New Yor |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL22293 |
| Series (1) |
| GSE185361 |
Rapid evolutionary dynamics of an expanding family of Drosophila meiotic drive factors and their hpRNA suppressors |
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| Relations |
| BioSample |
SAMN33260509 |
| SRA |
SRX19338860 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7039340_Dsim_RNA_rep1.Forward.bw |
121.4 Mb |
(ftp)(http) |
BW |
| GSM7039340_Dsim_RNA_rep1.Reverse.bw |
118.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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