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| Status |
Public on Jul 28, 2021 |
| Title |
Input_Mettl3_KD |
| Sample type |
SRA |
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| Source name |
cell culture
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| Organism |
Drosophila melanogaster |
| Characteristics |
antibody: Input Sample Pol II & Ser2P
cell type: Embryonic
cell line: S2R+ Cells
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| Treatment protocol |
dsRNA was transfected in S2R+ cells by serum starvation for 6 hours. The treatment was repeated two times and cells were harvested 5 days after the first treatment.
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| Growth protocol |
Drosophila S2R+ cells were cultured in Schneider Cell’s Medium (GIBCO, Cat No-21720) supplemented with 10% FBS and 2% Penicillin/Streptomycin
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
S2R+ cells were fixed with 1% formaldehyde for 3 min at room temperature, and harvested in SDS buffer resuspended in RIPA buffer (140 mM NaCl, 10 mM Tris-HCl [pH 8.0], 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% DOC), and lysed by sonication. The lysate was cleared by centrifugation, and incubated with respective antibodies overnight at 4ºC. Antibody complexes bound to protein G beads, were washed once with 140mM RIPA, four times with 500mM RIPA, one with LiCl buffer and twice with T.E buffer for 10 minutes each at 4ºC.
After checking enrichment the recovered DNA was converted into libraries using NebNext Ultra DNA library preperation kit, following manufacturer's protocol
NebNext UltraII
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Input Sample Pol II & Ser2P, with Mettl3 depletion
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| 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.
After mapping reads were filtered for uniquely mapping reads using the XS flag.
UCSC compatible library normalised bigwig tracks were produced using samtools version 1.2, ucsc tools (bedGraphToBigWig from 04-11-2012) and bedtools (v. 2.25.0) and custom scripts
Genome_build: dm6 (Drosophila melanogaster)
Supplementary_files_format_and_content: Ctrl_HA.bw: Replicate averaged, input normalized bigwig file of Control HA
Supplementary_files_format_and_content: Ctrl_KD_PolII.bw: Replicate averaged, input normalized bigwig file for Pol II with LacZ depletion
Supplementary_files_format_and_content: Ctrl_KD_Ser2P.bw : Replicate averaged, input normalized bigwig file for Ser2P with LacZ depletion
Supplementary_files_format_and_content: Mettl14_HA_UCSC.bw : Replicate averaged, input normalized bigwig file of Mettl14HA
Supplementary_files_format_and_content: Mettl14_KD_PolII_ave.bw: Replicate averaged, input normalized bigwig file for Pol II with Mettl14 depletion
Supplementary_files_format_and_content: Mettl14_KD_Ser2P_ave.bw : Replicate averaged, input normalized bigwig file for Ser2P with Mettl14 depletion
Supplementary_files_format_and_content: Mettl3_HA.bw: Replicate averaged, input normalized bigwig file of Mettl3 HA
Supplementary_files_format_and_content: Mettl3_HA_Ime4KD.bw : Replicate averaged, input normalized bigwig file of Mettl3 HA, with Mettl3 depletion
Supplementary_files_format_and_content: Mettl3_HA_RNaseT1.bw: Replicate averaged, input normalized bigwig file of Mettl3 HA, with RNAseT1 treatment
Supplementary_files_format_and_content: Mettl3_KD_PolII.bw: Replicate averaged, input normalized bigwig file for Pol II with Mettl3 depletion
Supplementary_files_format_and_content: Mettl3_KD_Ser2P.bw: Replicate averaged, input normalized bigwig file for Ser2P with Mettl3 depletion
Supplementary_files_format_and_content: Ythdc1_HA.bw: Replicate averaged, input normalized bigwig file of Ythdc1 HA
Supplementary_files_format_and_content: Ythdc1_KD_PolII.bw: Replicate averaged, input normalized bigwig file for Pol II with Ythdc1 depletion
Supplementary_files_format_and_content: fl2d_HA.bw: Replicate averaged, input normalized bigwig file of fl2d HA
Supplementary_files_format_and_content: fl2d_KD_PolII.bw: Replicate averaged, input normalized bigwig file for Pol II with fl2d depletion
Supplementary_files_format_and_content: fl2d_KD_Ser2P.bw: Replicate averaged, input normalized bigwig file for Ser2P with fl2d depletion
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| Submission date |
Jan 24, 2020 |
| Last update date |
Jul 28, 2021 |
| Contact name |
Junaid Akhtar |
| E-mail(s) |
akhtar@uni-mainz.de
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| Organization name |
University of Mainz
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| Department |
Institute of Neurobiology and Developmental Biology
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| Street address |
Johannes-Joachim-Becherweg, 32
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| City |
Mainz |
| State/province |
Rheinland-Pflaz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
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| Platform ID |
GPL19132 |
| Series (2) |
| GSE144244 |
m6A RNA methylation regulates promoter proximal pausing of RNA Pol II (ChIP-Seq) |
| GSE144246 |
m6A RNA methylation regulates promoter proximal pausing of RNA Pol II |
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| Relations |
| BioSample |
SAMN13919717 |
| SRA |
SRX7628913 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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