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Sample GSM6934448

Query DataSets for GSM6934448

Status

Public on May 25, 2023

Title

ZNII_Control, replicate 3

Sample type

SRA

Source name

Third-instar larvae

Organism

Drosophila melanogaster

Characteristics

tissue: Third-instar larvae
chip antibody: anti-GFP (TP-401, Torrey Pines Biolabs)

Treatment protocol

None

Growth protocol

All animals were raised in a 25°C incubator.

Extracted molecule

genomic DNA

Extraction protocol

75 wandering third-instar larvae (3 biological replicates per genotype) were collected in a 2ml DNA LoBind Eppendorf tube and washed twice with 1ml 1X PBS. The larvae were homogenized in ice-cold lysis buffer (200ul 1X protease inhibitor cocktail, 250ul PMSF, 800ul 1X PBS, 1ul Tween 20) using a pellet pestle. The homogenized lysate was supplemented with 244.5ul of 11% formaldehyde to a final concentration of 1.8% and the samples were crosslinked for 15 minutes at room temperature on a rotator. Glycine was added to a final concentration of 500mM to quench the fixative on ice for 5 minutes at room temperature. The larval debris was pelleted at 1,000g for 3 minutes and the supernatant was removed. The pellet was resuspended in 1ml sonication buffer (0.5% SDS, 20mM Tris pH 8.0, 2mM EDTA, 0.5mM EGTA, 0.5mM PMSF, 1X protease inhibitor cocktail) and chromatin was fragmented to 300 – 500bp Bioruptor sonicator (UCD-200) for 20 cycles (30 seconds high frequency sonication, 1.5 sec pause) in a cold room. The sonicated material was pelleted at 10,000g for 10 minutes at 4°C, supernatant was collected, then fragment size was checked prior to immunoprecipitation. The sonicated chromatin was pre-cleared and incubated with anti-GFP antibody overnight (TP-401, Torrey Pines Biolabs) 4°C overnight. The immunoprecipitated chromatin was then collected with pre-washed Protein A agarose beads for 2 hours. The beads were sequentially washed with the following buffers: 1 low salt buffer wash (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCL pH 8.0, 150mM NaCl), 3 high salt buffer washes (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCL pH 8.0, 500mM NaCl), 1 LiCL wash (2mM EDTA, 20mM Tris-HCl pH 8.0, 0.25M LiCl, 1% NP-40) and 2 TE buffer washers before elution. Bound chromatin on beads was eluted twice at room temperature using 250ul of freshly prepared ChIP elution buffer (1% SDS, 100mM NaHCO3) for 15 minutes and reverse-crosslinked overnight. The eluates were then treated with RNase A and proteinase K prior to DNA extraction via phenol-chloroform extraction and ethanol precipitation. Libraries were made and sequenced at Novogene.
Libraries were made using NEBNext Ultra II DNA Library Prep Kit at Novogene

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Data processing

The quality of FASTQ files (raw reads) were checked using FastQC (version. 0.11.9) and trimmed with fastp (version 0.23.2). Trimmed FASTQ files were aligned to the Drosophila genome (dm6) using Bowtie2 (version 2.4.5) to generate bam files. Unmapped and low-quality reads were discarded from bam files (<=20 mapQuality) using BamTools. Duplicate reads were identified and removed from mapped reads using Picard MarkDuplicates (http://broadinstitute.github.io/picard/). Deeptools MultiBamSummary was used to determine reproducibility of ChIP-Seq reads. MACS2 (Version 2.2.7.1) was used to narrowPeaks against pooled control using default settings. MACS2 bedGraph pileups were used to generate bigWig using using bedgraph-to-bigWig (UCSC, version 357).
Assembly: dm6
Supplementary files format and content: bigWig of ZNII and Control.

Submission date

Jan 13, 2023

Last update date

May 25, 2023

Contact name

Gbolahan Bamgbose

Organization name

University of North Dakota

Department

Biomedical Sciences

Street address

501 N Columbia Rd