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| Status |
Public on Jun 04, 2019 |
| Title |
L3 larvae brain RNA-seq: female MLE9 mutant; replicate 3 |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Drosophila melanogaster |
| Characteristics |
developmental stage: L3 larvae (3rd Instar)
genotype: MLE9 mutant
Sex: female
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| Growth protocol |
Fruit flies (Drosophila melanogaster) were reared on standard cornflour-molasses medium. at 25°C, 70% relative humidity and 12 hrs dark/12 hrs light cycle. mle9, cn, bw/CyO flies (BDSC #5873) have been ordered from the Bloomington Drosophila Stock Center (BDSC) and rebalanced with the help of w; CyO, Act5C-GFP/If to generate the w; mle9, cn, bw/CyO, Act5C-GFP flies used in this study.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from embryos was extracted using the Quick RNA Kit (Zymo Research). RNA from dissected brains of larvae was isolated using the DirectZol Kit (Zymo Research). RNA was eluted in 25 ul of RNase-free water.
See our manuscript for full protocol of MAPCap library construction
MAPCap: custom strategy explained in the manuscript. RNAseq: Illumina TRUseq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Description |
RNAseq performed on brains extracted from L3 larvae.
RNAseq_DEseq2_female_KO_vs_male_KO.tsv; RNAseq_featureCounts.tsv
RNAseq_larvae_mle9_female_rep3
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| Data processing |
MAPCap reads were further demultiplexed using the barcodes present in R2 read. Barcodes and corresponding sample names are provided as MAPCap_embryo_barcodes.tsv and MAPCap_larvae_barcodes.tsv. No demultiplexing required for RNAseq
MAPCap: Paired-end FASTQ files were trimmed for adaptors using Trimmomatic 56 (v 0.3.7). Samples were de-multiplexed by icetea (v0.99, demultiplexFastq) using provided barcode information, and mapped to the dm6 genome using Rsubread 57 (v 1.22.3, mapping wrapper provided in icetea). For de-duplication, we consider all reads mapping to the same 5’-position and having the same random barcode as duplicates and only keep the first instance of each such alignment (using icetea - filterDuplicates). BigWigs were created using deepTools 58 (v3.0.2) bamCoverage and bamCompare, with the option `--offSet 1 --binSize 1`.
RNA-seq: data was processed using snakePipes RNA-seq pipeline 65 (v1.0.0 beta) using the options ` -m alignment,deepTools_qc --star_options --limitBAMsortRAM 60000000000 --outBAMsortingBinsN 30 dm6`. snakePipes performed alignment using STAR 66 (v2.6.1a), counting of reads on Ensembl GTF (release 79) via featureCounts 67 (v1.6.1), and quality-checks via deepTools 58 (v3.1.2). The gene-level counts obtained from featureCounts were then used for differential expression analysis via DESeq2 68 (v1.20.0).
Pipeline for processing MAPCap data is available at: https://github.com/vivekbhr/cage_pipeline
Pipeline for processing RNAseq data is available at: https://github.com/maxplanck-ie/snakepipes
Genome_build: dm6
Supplementary_files_format_and_content: bw, featureCounts, DESeq2 output
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| Submission date |
Jan 29, 2019 |
| Last update date |
Jun 04, 2019 |
| Contact name |
Asifa Akhtar |
| E-mail(s) |
[email protected]
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| Organization name |
Max Planck Institute of Immunobiology and Epigenetics
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| Department |
Chromatin Regulation
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| Lab |
Akhtar Lab
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| Street address |
Stuebeweg 51
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| City |
Freiburg |
| ZIP/Postal code |
79108 |
| Country |
Germany |
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| Platform ID |
GPL23323 |
| Series (1) |
| GSE125831 |
MAPCap enables high-resolution detection and differential expression analysis of transcription start sites |
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| Relations |
| BioSample |
SAMN10840394 |
| SRA |
SRX5306513 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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