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Sample GSM6266314

Query DataSets for GSM6266314

Status

Public on Oct 28, 2022

Title

KeaA-HA_IP_biol_rep_3

Sample type

SRA

Source name

anti-HA immunoprecipitated biological replicate 3

Organism

Aspergillus nidulans

Characteristics

tissue: mycellium
cell type: KaeA-HA
genotype: (kaeA::HA, A.f. pyrG), pyrG89; argB2; pabaB22, nkuA{delta}::argB; riboB7

Growth protocol

Mycellium was grown in liquid minimal medium.

Extracted molecule

genomic DNA

Extraction protocol

ChIP was conducted according to Boedi et al., 2012 (Meth. in Mol. Biol., vol. 944, p.221-236; Springer Protocols). 1% formaldehade for 15 min was used as crosslinking agent, mycelia were grinded in mortar, extracts were sonicated using Bioruptor 200 (Diagenode) with 2 min "on"/1 min "off"/power "high" settings for 25 min and anti-HA 12CA5 antibody was used for chromatine immunoprecipitation (Roche, #11583816001).
ChIP-seq libraries were prepared using KAPA HyperPrep Kit and KAPA Dual-Indexed adapters (Kapa Biosciences) with 10 cycles of amplification. The size distribution was assessed using the Agilent 2100 Bioanalyzer and High Sensitvity DNA kit. The concentration of the libraries was determined by qPCR using the Kapa Library Quantification kit (Kapa Biosciences) and Roche LC480II cycler. Sequencing was performed on the Illumina NovaSeq 6000 instrument using the NovaSeq 6000 S2 Reagent Kit (100 cycles) (Illumina), with 2 × 100 cycles pair-end reading mode.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NovaSeq 6000

Description

2x100 bp paired-end sequencing

Data processing

Raw reads were trimmed with Trimmomatic software (Bolger et al., 2014). All reads with quality less than 20 were trimmed. Reads shorter than 50 nucleotides were discarded.
Trimmed reads were mapped to the reference genome with the use of BWA (Li et al.., 2010).
Duplicated reads were removed with the use of Picard tool (http://broadinstitute.github.io/picard/).
Peak calling was performed in R software (Team and Core, 2013), with the use of packages – systemPipeR, CHIPpeakAnno, GenomicFeatures, ChIPseeker and Biostrings. All biological replicates (fastq files) were merged into one file for input and IP. Peak calling with input/IP sample was performed with the use of MACS2 (Zhang, Yong et al., 2008) callpeak algorithm.
Differential binding analysis between control and treatment was performed with the use of edgeR (Robinson et al., 2010) package.
Motifs discovery was performed with the use of MEME-ChIP (Machanick et al., 2011) algorithm. Analysis was performed against YEASTRACT_20130918 database (Teixeira, Miguel et al., 2006).
Assembly: Aspergillus_nidulans.ASM1142v1 reference (https://fungi.ensembl.org/index.html)
Supplementary files format and content: Excel output of peak calling input/IP from MACS2: JG_T_FR.bam_macs2_peaks.xls
Supplementary files format and content: BED output of peak calling input/IP from MACS2: JG_T_FR.bam_macs2_summits.bed
Supplementary files format and content: Fasta sequences for each peak from MAC2: JG_T_FR.bam_macs2_peaks.fasta
Supplementary files format and content: Excel otput of peaks annotation on reference genome from MACS2: JG_T_FR.bam_macs2_peaks_annotated.xlsx
Supplementary files format and content: Excel output of reads counting for each peak from MACS2: JG_T_FR.bam_macs2_peaks.countDF.xls
Supplementary files format and content: Excel output of differential binding analysis between input and IP from edgeR: JG_T_FR.bam_macs2_peaks.edgeR.xls
Supplementary files format and content: TSV output from Interproscan for annotation with GO, Pfam and KEGG databases: JG_T_FR.bam_macs2_peaks_GO_PFAM_KEGG_annotated.tsv

Submission date

Jun 24, 2022

Last update date

Oct 28, 2022

Contact name

Agnieszka Dzikowska

E-mail(s)

adzik@igib.uw.edu.pl

Organization name

University of Warsaw

Department

Biology