GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM4910644

Query DataSets for GSM4910644

Status

Public on Oct 13, 2021

Title

C_H3K36me3.ctrl.st5.rep2

Sample type

SRA

Source name

embryos

Organism

Drosophila melanogaster

Characteristics

genotype: t180
timepoint: stage5
assay: ChIP-seq
chip antibody: H3K36me3 (Abcam, Cat. No. ab9050)

Extracted molecule

genomic DNA

Extraction protocol

ChIP-seq experiments were performed as in Zenk et al., 2017. Briefly, nuclei were extracted in lysis buffer (140 mM NaCl, 15 mM HEPES [pH 7.6], 1 mM EDTA, 0.5 mM EGTA, 1 % TritonX100, 0.5 mM DTT, 0.1 % Sodium Deoxycholate, 10 mM Sodium Butyrate, 1X Protease Inhibitors) and subsequent ultrasound treatment (Covaris E220, 45 sec, peak power 75, duty factor 10, cycles burst 200). Fixed chromatin was then sheared using Covaris E220 (900 sec, peak power 140, duty factor 5, cycle burst 200) and precleared overnight. Each sample was incubated with the relevant antibody and concentration for at least 4 hrs at 4°C. Samples were then washed, eluted and decrosslinked overnight by incubation at 65°C. Next, samples were treated with RNaseA(50mg/mL final concentration) for 30min at 37°C and ProteinaseK (200mg/mL, final) for 3 hrs at 56°C.
Libraries were prepared according to manufacturer’s instructions using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Libraries were quality controlled by capillary electrophoresis on the Fragment Analyzer system (Advanced Analytical).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

processed data file:
h3k36me3.subtract_input.ctrl.mean.bw

Data processing

Raw short read sequencing was processed uniformly using default parameters of snakepipes-v2.0.2 (Bhardwaj et al., 2019)). ChIP-seq samples were processed with the DNA-mapping workflow (–trim, --dedup, –properPairs), followed by assay-specific workflow, i.e. ChIP-seq.
Differential Pol II occupancy was determined using the method described in Blythe and Wieschaus, 2015. In brief, reads were countered per transcript using featureCounts (-t transcript -g transcript_id -f -O -Q 3 --primary -s 0). Reads were counted multiple times if they overlap several isoforms. Then, per transcript read counts were processed with edgeR (Robinson et al., 2010) to compute differential Pol2 occupancy. Like in the published method, transcripts spanning less than 125 nucleotides were discarded and only transcripts with read counts across replicates sum more that 10 reads were considered.
Genome_build: dm6, ensembl 96
Supplementary_files_format_and_content: Supplementary files are bigwigs contains library size and input corrected bigwig tracks and the promoter quantification of the Rpb3 (pol2) ChIP-seq.

Submission date

Nov 16, 2020

Last update date

Oct 13, 2021

Contact name

Nicola Iovino

E-mail(s)

[email protected]

Organization name

MPI of Immunobiology and Epigenetics

Street address

Stübeweg 51