 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 20, 2022 |
| Title |
OreR, 2to3, mnase, 2 |
| Sample type |
SRA |
| |
|
| Source name |
embryo
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryo
genotype: Wild-type (Oregon-R)
developmental stage: 2-3 hours after egg laying
|
| Treatment protocol |
All embryos were dechorionated with 50% bleach for 2 minutes, rinsed with water, and fixed with 1.8% formaldehyde while vortexing for 15 minutes. Fixation was quenched with PBT-glycine (125 mM glycine in PBT) and vortexing for 2 minutes. Embryos were hand-sorted based on morphology in ice-cold PBT and then immediately used in MNase-seq experiments.
|
| Growth protocol |
All embryos were collected from population cages using apple juice plates with yeast paste, following two pre-clearings. For MNase experiments, embryos were collected for 1 hour and aged for 2 hours at 25°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each MNase digestion, 100 hand-sorted 2-3 h AEL Drosophila embryos were used. Nuclei were extracted by douncing in PBS with 0.1% IGEPAL CA-630. The nuclei were harvested by centrifugation and resuspended gently in MNase Digestion Buffer (PBS with 0.1% Triton X-100 and 1 mM CaCl2). MNase digestion was performed with 100 U MNase (NEB, M0247S) for 30 minutes at 37°C. The reaction was stopped with 20 mM EGTA. The nuclei were treated with 50 µg/ml RNase A (Thermo Scientific, EN0531) for 1 hour at 37 °C and 1000 rpm, and subsequently incubated overnight at 65 °C and 1000 rpm with 200 µg/ml Proteinase K (Invitrogen, 100005393) and 0.5% SDS for reverse cross-linking. DNA was extracted using phenol-chloroform (VWR, K169).
MNase-seq libraries were constructed from 10 ng purified DNA using the High Throughput Library Prep Kit from KAPA Biosystems (KK8234) according to the manufacturer’s instructions.
MNase-seq for nucleosome occupancy using standard Illumina protocols.
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
MNase-seq paired-end sequencing reads were aligned using bowtie2 (v.2.3.5.1) to the Drosophila melanogaster genome assembly dm6.
Aligned MNase-seq BAM files were deduplicated based on unique fragment coordinates and filtered to contain fragment lengths no greater than 600 bp.
Genome-wide fragment coverage was calculated and saved in BigWig format.
Assembly: UCSC dm6
Supplementary files format and content: BigWig
|
| |
|
| Submission date |
Nov 27, 2022 |
| Last update date |
Dec 23, 2022 |
| Contact name |
Kaelan Joseph Brennan |
| E-mail(s) |
KBrennan@stowers.org
|
| Phone |
8125281836
|
| Organization name |
Stowers Institute for Medical Research
|
| Lab |
Zeitlinger
|
| Street address |
1000 East 50th Street
|
| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE218850 |
Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation [Mnase-seq] |
| GSE218852 |
Chromatin accessibility in the Drosophila embryo is determined by transcription factor pioneering and enhancer activation |
|
| Relations |
| BioSample |
SAMN31886423 |
| SRA |
SRX18393605 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6757767_orer_mbt_100u_mnase_2.bw |
249.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
