|
| Status |
Public on Nov 27, 2019 |
| Title |
Wagner_Dmel_DL1_IntS11dep+WTrescue_RNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
Drosophila DL1 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: DL1 cells
treatment: IntS11 RNAi
chip antibody: NA
gender: N/A
|
| Treatment protocol |
IntS11 RNAi
RNA-seq: 3 biological replicates were generated for Control (B-galactosidade treated) in Flag-tagged eGFP control line transgenes or IntS11-depletion (treated with double stranded RNA for IntS11) in Flag-tagged IntS11WT, E203Q mutant, and the eGFP control DL1 cell line transgenes. RNAi treatments were carried for 60h along with a final concentration of 100 μM CuSO4 to induce transgene expression. ATAC-seq: 3 biological replicates using untreated S2 cells were generated.
|
| Growth protocol |
FLAG-tagged IntS11WT Drosophila DL1 cells were cultured at 25°C in Schneider’s Drosophila medium (Thermo Fisher Scientific 21720024), supplemented with 10% (v/v) fetal bovine serum (HyClone SH30910.03), 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific 15140122), and 1% (v/v) L-glutamine (Thermo Fisher Scientific 35050061). RNAi treatments were carried for 60h along with a final concentration of 100 μM CuSO4 to induce transgene expression. For all experiments, cells were harvested at a consistent cell density of 4-6x106 cells/ml.
Drosophila S2 cells from the DGRC were grown in Shields and Sang M3 (Sigma) media supplemented with bactopeptone (BD Biosciences), yeast extract (Sigma) and 10% FBS (Thermo Fisher Scientific). Drosophila DL1 cells were cultured at 25°C in Schneider’s Drosophila medium (Thermo Fisher Scientific 21720024), supplemented with 10% (v/v) fetal bovine serum (HyClone SH30910.03), 1% (v/v) penicillin-streptomycin (Thermo Fisher Scientific 15140122), and 1% (v/v) L-glutamine (Thermo Fisher Scientific 35050061). For all experiments, cells were harvested at a consistent cell density of 4-6x106 cells/ml.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
total RNA was extracted with RNeasy Mini kit (Qiagen) following manufacturer's instructions.
NA
ATAC-seq experiments: genomic DNA was column extracted following manufacturer's instructions for Qiagen MinElute PCR purification kit. RNA-seq experiments: total RNA was extracted with RNeasy Mini kit (Qiagen) following manufacturer's instructions.
RNA-seq experiments: Click-Seq Total RNA (Jaworski et al. MiMB 2018), with Oligo-d(T) bead selection (NEB) from Drosophila DL1 cells treated with Bgal or IntS11 RNAi
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| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Base calling and generation of FASTQ files performed using standard CASAVA pipeline for HiSeq runs, bcl2fastq2 for NextSeq
ATAC-seq read pairs containing one or more members with mean quality score <20 were filtered. RNAseq read pairs containing one or more members with mean quality score <20 filtered and trimmed to 50nt
ATAC-seq mapped using bowtie 1.2.2 retaining uniquely mapped pairs only, allowing 2 mismatches. RNAseq mapped to reference using STAR 2.5.2b, in a strand-specific manner
ATAC-seq bedGraphs were generated by filtering duplicates and fragments of length >20 and <150, and determining counts of fragment centers in 25 nt bins tiling the genome, using custom scripts. RNAseq strand-specific coverage tracks were normalized with custom scripts using factors determined by DESeq2 v1.18.1 based on DESeq2 normalization factors and combined with unionBedGraphs and custom scripts (mean count).
Genome_build: dm3
Supplementary_files_format_and_content: ATAC-seq: bedGraph containing combined count of fragment centers for all replicates. RNAseq: bigWig containing combined mean normalized coverage of all replicates for a given treatment.
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| |
|
| Submission date |
Jul 03, 2019 |
| Last update date |
Nov 28, 2019 |
| Contact name |
Karen Adelman |
| E-mail(s) |
karen_adelman@hms.harvard.edu
|
| Organization name |
Harvard Medical School
|
| Department |
Biological Chemistry and Molecular Pharmacology
|
| Street address |
45 Shattuck St. LHRRB-201a
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL19132 |
| Series (1) |
| GSE114467 |
The Integrator complex terminates promoter-proximal transcription at protein-coding genes |
|
| Relations |
| BioSample |
SAMN12213642 |
| SRA |
SRX6404821 |
