|
| Status |
Public on Jul 28, 2021 |
| Title |
ime4_t2_R1 |
| Sample type |
SRA |
| |
|
| Source name |
cell culture
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: Embryonic
cell line: S2R+ Cells
|
| Treatment protocol |
dsRNA was transfected in S2R+ cells by serum starvation for 6 hours. The treatment was repeated three times and cells were harvested 7 days after the first treatment.
|
| Growth protocol |
Drosophila S2R+ cells were cultured in Schneider Cell’s Medium (GIBCO, Cat No-21720) supplemented with 10% FBS and 2% Penicillin/Streptomycin
|
| Extracted molecule |
total RNA |
| Extraction protocol |
5,6-dichlorobenzimidazole 1-_-d-ribofuranoside (DRB) from Sigma (D1916) was used at a final concentration of 300_M, dissolved in water, for 5 hours. 4-thiouridine (4sU) was purchased from Sigma (T4509) and used at a final concentration of 100 _M. Control and Mago KD was performed as described before. All the samples were labeled for 8 minutes with 4-thiouridine, and transcription was allowed to proceed after DRB removal for 0, 2, 8 and 16 minutes along with one non-DRB treated control.A total of 100 to 130 _g RNA was used for the biotinylation reaction. 4sU-labeled RNA was biotinylated with EZ-Link Biotin-HPDP (Pierce), dissolved in dimethylformamide (DMF) at a concentration of 1 mg/mL. Biotinylation was done in labeling buffer (10 mM Tris pH 7.4, 1 mM EDTA) and 0.2 mg/mL Biotin-HPDP for 2 h with rotation at room temperature. Two rounds of chloroform extractions removed unbound Biotin-HPDP. RNA was precipitated at 20,000 g for 20 min at 4¡C with a 1:10 volume of 5M NaCl and an equal volume of isopropanol. The pellet was washed with 75% ethanol and precipitated again at 20,000 g for 10 min at 4¡C. The pellet was left to dry, followed by resuspension in 100 _L RNase-free water. Biotinylated RNA was captured using Dynabeads MyOne Streptavidin T1 beads (Invitrogen). Biotinylated RNA was incubated with 50 _L Dynabeads with rotation for 15 min at 25¡C. Beads were magnetically fixed and washed with 1_ Dynabeads washing buffer. RNA-4sU was eluted with 100 _L of freshly prepared 100 mM dithiothreitol (DTT), and cleaned on RNeasy MinElute Spin columns (Qiagen).
Enriched nascent RNAs were converted to cDNA libraries with Drosophila Ovation Kit (Nugen) with integrated ribosomal depletion workflow
Nugen Ovation
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Biotin enrichment
Mettl3 (ime4) depleted nascent DRB-4SU-seq where the elongation inhibition was removed for 2 minutes
|
| Data processing |
De-multiplexing and fastq file conversion was performed using blc2fastq (v.1.8.4)
Mapping was performed using bowtie2 (v. 2.3.3) against ensembl release 84 of BDGP6 for Drosophila.
Genome_build: dm6 (Drosophila melanogaster)
|
| |
|
| Submission date |
Jan 24, 2020 |
| Last update date |
Jul 28, 2021 |
| Contact name |
Junaid Akhtar |
| E-mail(s) |
akhtar@uni-mainz.de
|
| Organization name |
University of Mainz
|
| Department |
Institute of Neurobiology and Developmental Biology
|
| Street address |
Johannes-Joachim-Becherweg, 32
|
| City |
Mainz |
| State/province |
Rheinland-Pflaz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE144245 |
m6A RNA methylation regulates promoter proximal pausing of RNA Pol II (DRB-4sU-seq) |
| GSE144246 |
m6A RNA methylation regulates promoter proximal pausing of RNA Pol II |
|
| Relations |
| BioSample |
SAMN13919736 |
| SRA |
SRX7628928 |
