GSM7977863: IgG; Drosophila melanogaster; OTHER - SRA

SRX22906606: GSM7977863: IgG; Drosophila melanogaster; OTHER
1 ILLUMINA (Illumina NovaSeq 6000) run: 19.2M spots, 3.9G bases, 1.1Gb downloads

External Id: GSM7977863_r1

Submitted by: Icahn School of Medicine at Mount Sinai

Study: Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis [CUT&RUN]

PRJNA1053400 • SRP478320 • All experiments • All runs

show Abstracthide Abstract

Genome organization can regulate gene expression and promote cell fate transitions. The differentiation of germline stem cells (GSCs) to oocytes in Drosophila involves changes in Genome organization mediated by heterochromatin and the nuclear pore complex (NPC). Heterochromatin represses germ-cell genes during differentiation and NPCs anchor these silenced genes to the nuclear periphery, maintaining silencing to allow for oocyte development. Surprisingly, we find that genome organization also contributes to NPC formation, mediated by the transcription factor Stonewall (Stwl). As GSCs differentiate, Stwl accumulates at boundaries between silenced and active gene compartments. Stwl at these boundaries plays a pivotal role in transitioning germ-cell genes into a silenced state and activating a group of oocyte genes and Nucleoporins (Nups). The upregulation of these Nups during differentiation is crucial for NPC formation and further genome organization. Thus, crosstalk between genome architecture and NPCs is essential for successful cell fate transitions. Overall design: Sequencing of duplicate samples after performing CUT&RUN on Bam GKD ovaries (enriched for undifferentiated stages), WT (differentiated stages) and stwl GKD ovaries for the transcription factor Stwl, the boundary element BEAF as well as histone marks: H3K4me3, H3k27ac, H3k9me3, H3k27me3 and H3k4me1

Sample: IgG

SAMN38859345 • SRS19875512 • All experiments • All runs

Organism: Drosophila melanogaster

Library:

Name: GSM7977863

Instrument: Illumina NovaSeq 6000

Strategy: OTHER

Source: GENOMIC

Selection: other

Layout: PAIRED

Construction protocol: PBS was removed and the samples were permeabilized in 1mL of Permeabilization Solution (PBST+1% Triton-X) rotating in RT for 1 hour. Samples were then incubated overnight at 4°C in primary antibody dilutions in freshly prepared BBT+ buffer (PBST + 1% BSA + 0.5 mM Spermidine + 2 mM EDTA + 1 large Roche complete EDTA-free tablets). Primary antibody was replaced with BBT+ buffer and quickly washed twice. Samples were then incubated in ~700 ng/ml of pAG-MNase in BBT+ buffer rotating for 4 hours at 25°C. Samples were then quickly washed twice in wash+ buffer (20 mM HEPES pH7.5 + 150 mM NaCl + 0.1% BSA + 0.5 mM Spermidine + 1 large Roche complete EDTA-free tablets in water). Samples were resuspended in 150 μl Wash+C (wash+ + 100 mM CaCl2) and incubated for 45 minutes on nutator at 4°C. The cleavage reaction was terminated by addition of 150 μl StopR (NaCl final 200 mM + EDTA final 20 mM + 100μg/mL RNaseA) and incubating the sample at 37˚C for 30 minutes. Samples were then centrifuged at 16,000 x g for 5 minutes and 300 μl of the supernatant was collected for DNA discovery. To the supernatant, 2 μL 10% SDS and 2.5 μL of 20 mg /mL Proteinase K was added and incubated at 50°C for 2 hours. Half of this was kept as a backup and half was used in bead cleanup. 20 μL AmpureXP bead slurry and 280 μL MXP buffer (20% PEG8000 + 2.5 M NaCl + 10 mM MgCl2 in water) was added to the sample and mixed thoroughly followed by 15 minutes incubation at RT. The beads were separated by magnet and supernatant was discarded. The beads were carefully washed with 80% ethanol for 30 seconds, while on the magnetic stand and air dried for 2 minutes. The beads were then resuspended in 10 μL DNase free water. The samples from CUT&RUN assay were used for library preparation using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (E7645, E7103) and adaptor ligated DNA were prepared without size selection. The samples from CUT&RUN assay were used for library preparation using NEBNext® Ultra™ DNA Library Prep Kit for Illumina® (E7645, E7103) and adaptor ligated DNA were prepared without size selection.

Runs: 1 run, 19.2M spots, 3.9G bases, 1.1Gb

Run	# of Spots	# of Bases	Size	Published
SRR27228099	19,217,624	3.9G	1.1Gb	2024-05-29

ID:: 30967451

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