 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Nov 23, 2023 |
| Title |
Tollrm9rm10 2to4h ChIP Input Replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Embryos
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Tollrm9/rm10 mutant
developmental stage: Early embryonic stage (2-4 h AEL)
antibody: N/A
spike-in: N/A
|
| Treatment protocol |
Formaldehyde fixation and chromatin preparation were performed essentially as described in 'Blythe, S.A., and Wieschaus, E.F. (2015). Zygotic genome activation triggers the DNA replication checkpoint at the midblastula transition. Cell 160, 1169-1181.'. Toll mutant embryos (2-4 h AEL) were dechorionated in dilute bleach, rinsed thoroughly with embryo wash buffer (PBS, 0.1% Triton X-100) and crosslinked in a mixture of 2 ml fixation buffer (PBS, 0.5% Triton X-100) and 6 ml heptane with 100 μl of 37% formaldehyde (Sigma-Aldrich, F8775) for 15 min at room temperature. Crosslinking was quenched by the addition of 125 mM glycine, embryos were washed (PBS, 0.5% Triton X-100), snap frozen in liquid nitrogen and stored at -80 degrees Celsius.
|
| Growth protocol |
Toll mutant (from gd7, Tollrm9/rm10 and Toll10B) embryos were collected from flies kept on potato-mash agar food and maintained at 25 degrees Celsius. Embryos were collected on apple juice plates with fresh yeast and aged at 25 degrees Celsius until they reached the range of hours after egg laying (AEL) for the required developmental stage.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
To prepare chromatin, embryos were homogenized in a glass dounce homogenizer by 20 strokes with a tight pestle in A1 buffer (15 mM HEPES pH 7.6, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 0.5 mM DTT, 0.5% Triton X-100 and protease inhibitor tablets (Roche)), centrifuged at 3500 g for 5 min at 4°C and the supernatant discarded. The remaining nuclear pellet was resuspended in 200 μl of sonication buffer (15 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA pH 8.0, 0.5 mM EGTA pH 8.0, 0.1% sodium deoxycholate, 1% Triton X-100 and protease inhibitor tablets (Roche)) supplemented with 0.5% SDS and 0.2% n-lauroylsarcosine and sonicated using a Bioruptor (Diagenode) with high power settings to obtain an average fragment size distribution of 200-500 bp, Sonicated chromatin was centrifuged at 13,000 rpm for 10 min at 4°C and diluted 5-fold in sonication buffer to reduce the concentration of detergents. Immunoprecipitations (IPs) were performed with 10 μg rabbit anti-CBP (homemade) Chromatin was incubated with anti-CBP overnight at 4°C and equal amounts of Protein A and G Dynabeads (Invitrogen), pre-blocked with BSA (1 mg/ml), were incubated with the samples for 4 h at 4°C. Chromatin corresponding to 10% of the amount in each IP was withdrawn to serve as an input for qPCR. Samples were subjected to 10 min washes with Wash A (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 0.1% SDS, 0.1% Sodium Deoxycholate and 1% Triton X-100), Wash B (Wash A adjusted to 500 mM NaCl), Wash C (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCl, 0.5% Sodium Deoxycholate and 0.5% IGEPAL CA-630) and Tris-EDTA (TE) buffer. Beads were resuspended in 100 μl TE and treated with RNase A (20 μg/ml) at 55°C for 30 min before SDS (to 0.75%) and Tris-HCl (to 50mM) were added and crosslinks reversed at 65°C for overnight. Eluted ChIP DNA was treated with Proteinase K at 55°C for 2 h and purified with the ChIP DNA Clean & Concentrator kit (ZymoResearch, D5205).
ChIP-sequencing was performed using 2-5 ng of ChIP DNA in biological duplicates. Libraries were prepared with the NEBNext® Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) and single-end (1 x 75 bp) sequenced on the Illumina NextSeq 550 platform at the BEA core facility, Karolinska Institutet, Stockholm.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
Sequenced ChIP Input DNA
|
| Data processing |
ChIP-seq reads from each sequenced library were mapped to the Drosophila melanogaster (dm6) genome assembly using Bowtie2 with the default parameters. RPKM normalized ChIP-seq coverage tracks were generated using the deepTools (v.3.5.1) package (default parameters with binSize = 2 (bases), normalizeUsing RPKM). Bigwig files of the mean RPKM were produced using the deepTools 'bigwigCompare' tool.
Assembly: dm6
Supplementary files format and content: bigwig for coverage
|
| |
|
| Submission date |
Aug 15, 2022 |
| Last update date |
Nov 23, 2023 |
| Contact name |
Mattias Mannervik |
| E-mail(s) |
mattias.mannervik@su.se
|
| Organization name |
Stockholm University
|
| Department |
Dept. of Molecular Biosciences
|
| Street address |
Svante Arrhenius väg 20C
|
| City |
Stockholm |
| ZIP/Postal code |
106 91 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL22106 |
| Series (2) |
| GSE211219 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network [ChIP-Seq] |
| GSE211220 |
Tissue-specific RNA Polymerase II promoter-proximal pause release and burst kinetics in a Drosophila embryonic patterning network |
|
| Relations |
| BioSample |
SAMN30311156 |
| SRA |
SRX17064438 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6456232_Tollrm9rm10_2to4h_ChIP_Input_R1.bw |
152.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
