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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 16, 2023 |
| Title |
NC, 32C, chromatin input control, rep1 |
| Sample type |
SRA |
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| Source name |
FACS purified germ cell
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: FACS purified germ cell
Sex: female
developmental stage: 32C NC
genotype: otu-Gal4, mata-gal4, nos-gal4, UASz-tdTomato
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| Growth protocol |
flies were grown on standard food plus wet yeast paste for 3-6 days
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chip-seq was carried out as described in Deluca et al (2020). Since we worked with cells with various ploidy levels spanning from 2C to 512C, we standardized input to the number of genomes rather than the number of cells. Specifically, for each IP, the equivalent of 500,000 2C sorted nuclei (i.e. 1 million genomes) were mixed with a specific amount of fixed mouse 3T3 cells as a spike-in (the same amount across samples). Mixed samples were then sonicated to acquire mono-nucleosome fragments: nuclei were resuspended in 100 µl of Nuclei Resuspension Buffer (all recipes at end), and were sonicated in a Bioruptor Pico instrument with 22 cycles of 30 s on, 30 s off. Fragment sizes were confirmed with the Bioanalyzer (Agilent). Subsequently, 900 µl of Dilution Buffer was added to the 100 ul of mononucleosome fragments, then 1% of each sample was set aside to be used for input controls. To prepare antibody-conjugated beads, 10 µl of antibody (either H3K27ac, H3K27me3, or H3K9me3) was preincubated with 25 µl of a 1:1 mix of proteinA:proteinG dynabeads, then washed twice with PBS + 0.02% Tween 20 (PBST). To bind mononucleosome fragments that harbored the target antigen, we combined antibody-conjugated beads with the chromatin extract(s) on a rocker at 4°C overnight. The next day, the beads were washed twice for 15 min each with Wash buffer A, Wash buffer B, Wash buffer C, and TE buffer. Chromatin was eluted and reverse-crosslinked by incubating at 65°C overnight with Direct Elution Buffer (DEB). For input samples (set aside earlier), cross links were reversed by adding NaCl to 300 mM, then adding DEB to equalize the volume between inputs and IPs, then incubating at 65°C overnight. All samples were then incubated for 30 min at 37°C with 0.3 mg/ml RNAse A, and subsequently for 2 hr at 55°C with 0.6 mg/ml proteinase K. DNA was then extracted with phenol:chloroform, precipitated with NaAc/ethanol, washed with 70% ethanol wash, and resuspended samples in 10 µl water and of which all 10 µl was used for library preps. Recipes: Nuclei Resuspension Buffer (50 mM TrisHCl pH 8.0, 10 mM EDTA, 1% SDS, and proteinase inhibitor cocktail (Roche)). Dilution Buffer (15 mM TrisHCl pH 8.0, 1 mM EDTA, 1% Triton X-100, 150 mM NaCl). Wash buffer A (20 mM TrisHCl pH8.0, 2 mM EDTA, 0.1% SDS, 1% Triton X100, 150 mM NaCl), Wash buffer B (20 mM TrisHCl pH8.0, 2 mM EDTA, 0.1% SDS, 1% Triton X100, 500 mM NaCl), Wash buffer C (10 mM TrisHCl pH8.0, 1 mM EDTA, 1% NP40, 1% Sodium deoxycholate, 0.25M LiCl), TE buffer (10 mM TrisHCl pH8.0, 1 mM EDTA). Direct Elution Buffer (DEB) (10 mM TrisHCl pH 8.0, 300 mM NaCl, 5 mM EDTA, 0.5% SDS).
The Illumina sequencing library was prepared with Takara Bio / Rubicon ThruPLEX DNA-seq kit and sequenced on an Illumina NextSeq 500 to yield 75 bp, single-end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
H3K27ac ChIP-seq of FACS-purified Drosophila melanogaster germ cells
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| Data processing |
Reads were mapped with Bowtie2 (2.3.2). SAM output was filtered for MAPQ (>= 30) and to retain only reads on the major chromosome scaffolds (2L, 2R, 3L, 3R, 4, X and Y), sorted, converted to BAM, and indexed with SAMtools (1.6). A BigWig file was generated from the sorted filtered BAM using DeepTools (3.1.3; -p max -bs 1 --effectiveGenomeSize 127324632 --normalizeUsing CPM).
Assembly: Genome and Gene Annotation Set: BDGP6.22.Ensembl.98 (dm6, Ensembl release 98, NCBI: GCA_000001215.4). For samples with mouse cell spike-ins, a "hybrid genome assembly" was constructed by combining the BDGP6.22.Ensembl.98 dm6 Drosophila genome assembly with the mm10 mouse genome assembly from which 'hybrid genome' bowtie2 indexes were constructed.
Supplementary files format and content: RNAseq: STAR-RSEM output of estimated expression ; ChIPseq: bigWig ; HMM state paths: bedGraph with states 1 (depleted), 2 (no-change), and 3 (enriched). HMM gene annotations: BED-like tab-sepatated file with description in column names ;
Supplementary files format and content: AllDataTable.csv: aggregated data table of processed data of ATACseq, H3K27ac ChIPseq, H3K27me3 ChIPseq, and RNAseq from GSC to stage 9 nurse cells (NCs)
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| Submission date |
Apr 18, 2023 |
| Last update date |
Aug 16, 2023 |
| Contact name |
Allan C Spradling |
| E-mail(s) |
spradling@carnegiescience.edu
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| Phone |
(410) 246-3015
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| Organization name |
Carnegie Institution for Science
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| Department |
HHMI Lab at Embryology
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| Lab |
Spradling
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| Street address |
3520 San Martin Drive
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| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21218 |
| Country |
USA |
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| Platform ID |
GPL19132 |
| Series (1) |
| GSE229943 |
Chromatin and gene expression changes during female Drosophila germline stem cell development. |
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| Relations |
| BioSample |
SAMN34232807 |
| SRA |
SRX20000271 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7181652_NC_032C_input.chr.bw |
240.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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