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| Status |
Public on Jul 17, 2024 |
| Title |
elav>Pnt-Dam DamID-Seq biol rep 3 |
| Sample type |
SRA |
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| Source name |
third instar larval eye disc
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| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: third instar larval eye disc
cell type: photoreceptors
genotype: elav-GAL4; UAS-Pnt-Dam
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To prepare genomic DNA for DamID, 100 pairs of eye discs were homogenized in 200 µl ice-cold 0.1M Tris pH 9/0.1M EDTA/1%SDS (solution A). Another 300 µl of ice-cold solution A was added and the samples were incubated for 25 min at 70°C. 70 µl of 8 M potassium acetate was added and samples were incubated on ice for 30 min. The samples were centrifuged at 4°C at 14000 rpm for 15 min and the supernatants were extracted with an equal volume of phenol-chloroform solution (Sigma-Aldrich 77618 phenol-chloroform-isoamyl alcohol mixture 49.5:49.5:1), followed by a chloroform extraction. DNA was precipitated from the supernatant with 0.5 volume of isopropanol, incubated at room temperature for 5-10 min and centrifuged for 15 min at 4°C. The pellet was washed with 1 ml of 70% ethanol, air-dried and resuspended in 50 µl water overnight. To prepare RNA for RNA-Seq, RNA was extracted using Trizol (Invitrogen) followed by genomic DNA elimination and further purification with a Qiagen RNeasy Plus Micro kit. RNA quality and quantity was assessed using a Bio-analyzer 2100 (Agilent) prior to library preparation. To prepare single cells, eye discs were dissected into ice cold Rinaldini solution with 1.9 μM Actinomycin-D. Single cells were dissociated from eye discs mechanically by pipetting and enzymatically with Collagenase (100 mg/ml; Sigma-Aldrich #C9697) and Dispase (1 mg/ml; Sigma-Aldrich #D4818). Dissociated single cells were filtered and washed once with 0.05% Bovine Serum Albumin (BSA) in Rinaldini solution. Cells were resuspended in 0.05% BSA in Rinaldini solution to a concentration of 1000-1200 cells/μl. Only samples with over 95% viability (assayed with Hoechst propidium iodide) were used for scRNA-seq.
DamID libraries were prepared as described (Marshall, O. J., Southall, T. D., Cheetham, S. W. & Brand, A. H. Cell-type-specific profiling of protein-DNA interactions without cell isolation using targeted DamID with next-generation sequencing. Nat Protoc 11, 1586-1598 (2016).) RNA-Seq libraries were constructed using Illumina TruSeq Stranded mRNA (Cat #20020595), with 250 ng of total RNA as input, and 10 cycles of PCR amplification. scRNA-Seq library was prepared using the 10x Genomics Chromium Next GEM Single Cell 3’ Reagent Kit 3v31.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Basecalls performed using CASAVA
RNA-Seq reads were mapped using STAR guided by GTF and DamID reads were mapped using bowtie2
BAM to bedgraph performed using bedtools
bedgraph to bigwig performed using bedGraphToBigWig v4
Assembly: Dm6 (Drosophila melanogaster)
Supplementary files format and content: Big Wig (.bw)
Library strategy: DamID-Seq
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| Submission date |
Feb 21, 2024 |
| Last update date |
Jul 17, 2024 |
| Contact name |
Alireza Khodadadi-Jamayran |
| Organization name |
New York University, NYU Langone Medical Center
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| Department |
Division of Advanced Research Technologies (DART)
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| Lab |
Applied Bioinformatics Laboratories (ABL)
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| Street address |
550 1st Ave, MSB 304
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| City |
New York City |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platform ID |
GPL21306 |
| Series (1) |
| GSE256221 |
Synergistic activation by Glass and Pointed promotes neuronal identity in the Drosophila eye disc |
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| Relations |
| BioSample |
SAMN40012822 |
| SRA |
SRX23678542 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8089490_DamIDEp3_S15_RPM.bw |
74.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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