|
| Status |
Public on Apr 08, 2020 |
| Title |
GMR-GAL4_UAS-NERFIN-HA |
| Sample type |
SRA |
| |
|
| Source name |
L3 Drosophila eye-antennal disc
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: M{UAS-nerfin-1.ORF.3xHA.GW}ZH-86Fb
sequencing method: Omni-ATAC-seq
|
| Growth protocol |
All flies were raised and crossed at 25°C on a yeast based medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, ~10 eye-antennal discs were dissected and lysed using 50 µl of cold ATAC-Resupension Buffer (RSB) (see Corces et al. for composition) containing 0.1% NP40, 0.1% Tween-20 and 0.01% digitonin by pipetting up and down three times and incubating the cells for 3 min on ice. The lysis was washed out by adding 1 mL of cold ATAC-RSB containing 0.1% Tween-20 and inverting the tube three times.
Nuclei were pelleted at 500 RCF for 10 min at 4°C, the supernatant was carefully removed and nuclei were resuspended in 50 µl of transposition mixture (25 µl 2x TD buffer (see Corces et al. for composition), 2.5 µl transposase (100 nM), 16.5 µl DPBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-20, 5 µl H2O) by pipetting six times up and down, followed by 30 minutes incubation at 37°C at 1000 RPM mixing rate. After MinElute clean-up and elution in 21 µl elution buffer, the transposed fragments were pre-amplified with Nextera primers by mixing 20 µl of transposed sample, 2.5 µl of both forward and reverse primers (25 µM) and 25 µl of 2x NEBNext Master Mix (program: 72°C for 5 min, 98°C for 30 sec and 5 cycles of [98°C for 10 sec, 63 °C for 30 sec, 72°C for 1 min] and hold at 4°C). To determine the required number of additional PCR cycles, a qPCR was performed (see Buenrostro et al.83 for the determination of the number of cycles to be added). The final amplification was done with the additional number of cycles, samples were cleaned-up by MinElute and libraries were prepped using the KAPA Library Quantification Kit as previously described82. Samples were sequenced on a NextSeq500 High Output chip, with 50bp single-end reads.
Omni-ATAC-seq (Corces et al., 2017)
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Omni-ATAC-seq protocol as described by Corces et al. (2017)
|
| Data processing |
ATAC-seq reads were first cleaned for adapters using fastq-mcf. (ea-utils v1.12) and a list of sequencing primers. Cleaned reads (FastQC v0.1) were then mapped to the 3rd 2017 FlyBase release (D. melanogaster r6.16) genome using Bowtie2 (v2.2.5) with default parameters, with the single end option (to compare with the WT sample, which was single-end sequenced).
Sorted bam files were produced using using SAMtools (v1.2). Normalized bigwigs were generated using the Kent software (UCSC). Peaks were called on mapped reads using MACS2 (v2.1.2.1) with the following options: -g dm –nomodel –bdg -t Sample/Control -c Sample/Control (depending on whether we want to determine upregulated or downregulated peaks).
Genome_build: dm6
Supplementary_files_format_and_content: Bigwig files containing the normalized coverage profiles of each experiment.
|
| |
|
| Submission date |
Dec 06, 2019 |
| Last update date |
Apr 09, 2020 |
| Contact name |
Carmen Bravo González-Blas |
| E-mail(s) |
carmen.bravogonzalezblas@kuleuven.vib.be
|
| Organization name |
VIB-KU Leuven
|
| Department |
VIB-KU Leuven Center for Brain and Disease Research
|
| Lab |
Laboratory of Computational Biology
|
| Street address |
Herestraat 49
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL25244 |
| Series (2) |
| GSE141584 |
Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics [Omni-ATACseq] |
| GSE141590 |
Identification of genomic enhancers through spatial integration of single-cell transcriptomics and epigenomics |
|
| Relations |
| BioSample |
SAMN13499614 |
| SRA |
SRX7282928 |
