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Sample GSM7181657 Query DataSets for GSM7181657
Status Public on Aug 16, 2023
Title NC, 8C, H3K9me3 ChIP-seq, rep1
Sample type SRA
 
Source name FACS purified germ cell
Organism Drosophila melanogaster
Characteristics tissue: FACS purified germ cell
Sex: female
developmental stage: 8C NC
genotype: MTD-Gal4, UASz-tdTomato
Growth protocol flies were grown on standard food plus wet yeast paste for 3-6 days
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Description H3K9me3 ChIP-seq of FACS-purified Drosophila melanogaster germ cells
Data processing Reads were mapped with Bowtie2 (2.4.4). SAM output was filtered for MAPQ (>= 20), sorted, converted to BAM, and indexed with SAMtools (1.6). A genome-wide coverage bedGraph was computed with BEDtools (2.30.0). A BigWig file was generated using wigToBigWig from Kent Utils 420 (-clip). BEDtools was used to obtain coverage counts in 250 kb bins across the genome.
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)
 
Submission date Apr 18, 2023
Last update date Aug 16, 2023
Contact name Allan C Spradling
E-mail(s) [email protected]
Phone (410) 246-3015
Organization name Carnegie Institution for Science
Department HHMI Lab at Embryology
Lab Spradling
Street address 3520 San Martin Drive
City Baltimore
State/province Maryland
ZIP/Postal code 21218
Country USA
 
Platform ID GPL19132
Series (1)
GSE229943 Chromatin and gene expression changes during female Drosophila germline stem cell development.
Relations
BioSample SAMN34232802
SRA SRX20000276

Supplementary file Size Download File type/resource
GSM7181657_NC_8C_H3K9me3_HZ3-250kb.bedGraph.gz 15.4 Kb (ftp)(http) BEDGRAPH
GSM7181657_NC_8C_H3K9me3_HZ3.bw 52.3 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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