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Sample GSM4910666

Query DataSets for GSM4910666

Status

Public on Oct 13, 2021

Title

Input-RELACS.dom-kd.st5_rep2

Sample type

SRA

Source name

embryos

Organism

Drosophila melanogaster

Characteristics

genotype: t216
timepoint: stage5
assay: RELACS
chip antibody: none (input)

Extracted molecule

genomic DNA

Extraction protocol

RELACS protocol was performed as described previously by Arrigoni, et al. 2018. Briefly, embryos were thawed in RELACS lysis buffer (10 mM Tris-HCl [pH 8], 10 mM NaCl, 0.2% Igepal, 1x Protease inhibitor cocktail) and the nuclei were isolated by sonication using the NEXSON procedure(Arrigoni et al., 2016). To digest the chromatin, 25 µl of 10x CutSmart buffer (NEB), 2.5 µl 100x Protease inhibitor cocktail and 1 µl of CviKI-1 (50 U/µl, NEB R0710S) were added. The digestion reaction was incubated overnight at 20 °C. End repair and A-tailing was performed and customized adapters (Arrigoni et al., 2018) were ligated to the fragments. Once barcoded, the samples were pooled together. Chromatin was then sheared by sonication (Covaris E220, MicroTubes, 5min, peak power 105, duty factor 2, cycles burst 200). This chromatin was used for automated ChIP with the IP-Star Diagenode system. IPs and Inputs were decrosslinked and DNA was purified.
Libraries were prepared using the NEB Ultra II DNA Library Prep Kit for Illumina (E7645S and E6440) following the manufacturer’s instructions

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

Illumina HiSeq 3000

Data processing

Library strategy: RELACS ChIP-seq
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.

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