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| Status |
Public on Jun 23, 2023 |
| Title |
Dme_emb_orc4_14-17h_Rpb3_1 |
| Sample type |
SRA |
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| Source name |
Pol II ChIP in 14-17hrs Lola-I mutant ORC4 embryos Rep1
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: embryos
chip antibody: anti-RPB3 rabbit polyclonal, custom (GenScript)
developmental stage: 14-17h
genotype: Lola-I mutant:ORC4
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| Treatment protocol |
Embryos were dechorionated in 5% bleach for ~2-3 min, washed with water, fixed for 15 min shaking on a vortexer, speed 7 with 1.8% formaldehyde in heptane, washed with PBT-glycine and PBT, and frozen in liquid nitrogen until used.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq were performed as follows. ~100 mg embryos were used per ChIP, and 5 µg chromatin was used for tissue-specific ChIP-seq experiments. Fixed embryos were homogenized by douncing in an ice cold A1 buffer (15 mM HEPES (pH 7.5), 15 mM NaCl, 60 mM KCl, 4 mM MgCl, 0.5% Triton X-100, 0.5mM DTT, protease inhibitors) and A2 buffer (15 mM HEPES (pH 7.5), 140 mM NaCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.5% N-lauroylsarcosine, protease inhibitors) in a tissue grinder for 10-15 times in A1 and A2 buffer each. Then the sonication of the chromatin was performed with a Bioruptor Pico for four-five rounds of 30 s on and 30 s off cycles. The sonicated chromatin was cleared by centrifugation and the supernatant was used for ChIP. Chromatin was incubated with antibodies pre-bound to Dynal magnetic beads (IgA or IgG) overnight with end-to-end rotation at 4°C and washed with an ice cold RIPA buffer (50 mM HEPES (pH 7.5), 1mM EDTA, 0.7% sodium deoxycholate, 1% NP-40 (IGEPAL CA-630), 0.5M LiCl). Eluted, reverse cross-linked DNA was then purified using phenol-chloroform-isoamylalcohol phase separation and ethanol precipitation.
ChIP-seq libraries were prepared from 5-15 ng ChIP DNA or 100 ng input DNA according to the manufacturer’s instructions (NEBNext ChIP-Seq Library Prep kit).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
base calling, read filtering and demultiplexing were performed by Illumina CASAVA 1.8.2 with default settings.
For ChIP-seq samples, reads were aligned to the reference genome using bowtie v1.0.0, retaining only uniquely aligning reads with a maximum of 2 mismatches. Aligned reads were extended to each library's estimated insert size. Genome-wide coverage counts were calculated, and saved in BigWig format
Assembly: dm6
Supplementary files format and content: BigWig files contain pileup counts for each base
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| Submission date |
Apr 15, 2022 |
| Last update date |
Jun 23, 2023 |
| Contact name |
Julia Zeitlinger |
| E-mail(s) |
[email protected]
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| Organization name |
Stowers Institute
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| Street address |
1000 E 50th St
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| City |
Kansas City |
| ZIP/Postal code |
64110 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (2) |
| GSE200870 |
Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development [ChIP-seq] |
| GSE200875 |
Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development |
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| Relations |
| BioSample |
SAMN27597189 |
| SRA |
SRX14869069 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6045786_Dme_emb_orc4_14-17h_Rpb3_1.bw |
77.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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