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| Status |
Public on May 24, 2024 |
| Title |
Wing disc, K9R, H3K9me3, rep1 |
| Sample type |
SRA |
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| Source name |
wing disc
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: wing disc
cut&run antibody: H3K9me3 (Abcam #ab8898)
genotype: K9R
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| Growth protocol |
Stocks were maintained on standard cornmeal food at 25˚C. H3.2HWT and H3.2K9R larvae were cultured as previously described in Penke 2016. Briefly, one hundred fifty yw; ∆HisC, twi-Gal4/CyO virgin females and seventy-five yw; ∆HisC, UAS-2xEYFP/CyO; H3.2HWT/ H3.2HWT or H3.2K9R/ H3.2K9R males were put in a cage and allowed to lay eggs on a grape juice agar plates. GFP-positive L1 larvae were transferred to vials of standard food and allowed to grow at 25˚C. Wandering third instar (L3) GFP-positive larvae were used for experiments. HP1acontrol and HP1achromo larvae were cultured by establishing vials with ten w1118; Df(2L)BSC228/CyO, Tb, RFP virgin females and, to produce HP1acontrol, a single yw; +; + male, or a single w1118; HP1achromo/CyO, Tb, RFP male (Table S3). After three days, genomic DNA from sacrificed w1118; HP1achromo/CyO, Tb, RFP males was sequenced to confirm the genotype the HP1achromo allele, which exhibits occasional instability. The remaining females were flipped daily and non-tubby third larval instar wandering females were used for experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
A minimum of 20 wing imaginal discs per replicate were dissected from wandering female L3 larvae and used as input for CUT&RUN, as described previously (Ahmad 2018). The following antibody concentrations were used in CUT&RUN experiments: 1:100 anti-H3K9me3 (Abcam ab8898), 1:100 anti-HP1a (Developmental Studies Hybridoma Bank C1A9), 1:100 anti-GFP (Rockland 600-401-215), 1:100 anti-H3K27me3 (Cell Signal Technology #9733s), and 1:100 protein A/G-MNase. Using expression vector from Addgene (plasmid #123461), protein A/G-MNase was produced by the UNC Protein Expression and Purification Core using a published protocol (Meers 2019).
Libraries were prepared using Takara ThruPLEX DNA-Seq Kit (R400675) with Takara DNA Unique Dual Index Kit (R400665R, R400666R, R400667R, R400668R).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Fastq files for biological replicates were combined. Adapters were trimmed using bbmap version 38.71 and parameters ktrim=k, ref=adapters, rcomp=t, tpe=t, tbo=t, hdist=1, mink=11 for all fastq files (individual replicates and merged fastq files). Trimmed reads were aligned to the dm6 reference genome using bowtie2 version 2.3.4.1 and parameters --seed 123 -q --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 400. Sam files were then converted to bam files and reads were filtered using samtools version 1.10 such that only reads with a MAPQ greater than 5 were kept. Bam files were sorted using samtools and converted to bed files using bedtools version 2.26. Read depth was calculated and rpgc-normalized bedgraphs were generated from bed files using bedtools. Bedgraphs were converted to bigWigs using ucsctools version 320. Peaks were called from bed files using macs2 version 2.1.2 with parameters --nomodel, --seed 123. As a control for peak calling, we used sonicated genomic DNA.
Peak calls from negative control experiments were used to create a list of blacklisted peaks for each experimental antibody, which were filtered out of experimental data using the bedtools function intersect with option -v at the bam to bed file conversion step. Peaks called from IgG CUT&RUN were used in blacklists for all experiments. Peak calls from additional negative control experiments were combined with IgG peak calls to create specific blacklists for each experiment, as follows. For C1A9 HP1a CUT&RUN, only IgG peaks were blacklisted. For GFP-HP1a CUT&RUN, peaks called from anti-GFP CUT&RUN on yw wing imaginal discs, which lack the GFP epitope, were combined with IgG peaks. For H3K9me3 CUT&RUN, we examined the possibility that loss of the H3K9me3 epitope in H3.2K9R wings resulted in cross-reactivity of the H3K9me3 antibody with H3K27me3 due to similarity in the surrounding amino acids80. Comparison of H3K9me3 and H3K27me3 signals in H3.2HWT and H3.2K9R CUT&RUN showed evidence of cross-reactivity (Figure S3F-G). Therefore, we included peaks called from H3K27me3 CUT&RUN in the blacklist for H3K9me3 CUT&RUN. H3.2HWT H3K27me3 was used to blacklist H3.2HWT H3K9me3, H3.2K9R H3K27me3 was used to blacklist H3.2K9R H3K9me3, and yw H3K27me3 was used to blacklist HP1acontrol and HP1achromo H3K9me3 profiles.
Assembly: dm6
Library strategy: CUT&RUN
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| Submission date |
Jul 10, 2023 |
| Last update date |
May 24, 2024 |
| Contact name |
Daniel McKay |
| Organization name |
University of North Carolina at Chapel Hill
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| Street address |
250 Bell Tower Drive
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platform ID |
GPL21306 |
| Series (2) |
| GSE234277 |
Heterochromatic 3D genome organization is directed by HP1a and H3K9-dependent and independent mechanisms. |
| GSE236964 |
Heterochromatic 3D genome organization is directed by HP1a and H3K9-dependent and independent mechanisms[CUT&RUN] |
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| Relations |
| BioSample |
SAMN36390750 |
| SRA |
SRX20968559 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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