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| Status |
Public on Jun 23, 2023 |
| Title |
Gut_110394_INTACT_Lola_I_14-17h_2 |
| Sample type |
SRA |
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| Source name |
Lola-I ChIP in 14-17hrs INTACT embryos in Gut Rep2
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Gut
chip antibody: anti-Lola-I rabbit polyclonal, custom (GenScript)
developmental stage: 14-17h
genotype: wildtype
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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 |
Nuclei were isolated from INTACT embryos 14-17h AED by resuspending and douncing embryos in HBS buffer (0.125 M Sucrose, 15 mM Tris (pH 7.5), 15 mM NaCl, 40 mM KCl, 2 mM EDTA, 0.5 mM EGTA). The nuclei were further dissociated using syringe (22.5 gauge needle) 10 times. After washins with HBS buffer, nuclei were incubated with Dynabeads® M-280 Streptavidin beads( Invitrogen, # 11205D) for 30 minutes with end to end rotation at 4°C. A magnet was used to separate the bead bound nuclei, and the beads were washed thoroughly with HBS buffer. Purity of nuclei isolation was determined by staining nuclei with DAPI, and presence of free nuclei, unbound to beads, were used as indicators of contamination. Isolated nuclei were used for ChIP-seq. ChIP-seq was 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 NextSeq 500 |
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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 |
GPL19132 |
| Series (2) |
| GSE200869 |
Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development [ChIP-seq INTACT] |
| GSE200875 |
Lola-I is a promoter pioneer factor that establishes de novo Pol II pausing during development |
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| Relations |
| BioSample |
SAMN27597166 |
| SRA |
SRX14868965 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6045733_Gut_110394_INTACT_Lola_I_14-17h_2_R1.bw |
42.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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