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Sample GSM7230175 Query DataSets for GSM7230175
Status Public on Jan 30, 2024
Title His-Flag_ChIP
Sample type SRA
Source name Flp-In T-Rex HEK293
Organism Homo sapiens
Characteristics cell line: Flp-In T-Rex HEK293
genotype: WT with Flp-In locus modified fro expression of His-FLAG
chip antibody: anti-FLAG M2 (Sigma-Aldrich Cat#M8823)
Treatment protocol Expression of tagged proteins was induced by addition of tetracycline for 24 h before crosslinking with 1.5 mM EGS for 10 min at room temperature and 1% PFA for 45 min at 4°C.
Growth protocol HEK293 Flp-In T-Rex cell lines expressing GPATCH4-His-FLAG or the His-FLAG tag alone were grown at 37°C in 5% CO2 in high glucose DMEM supplemented with 10% foetal calf serum .
Extracted molecule genomic DNA
Extraction protocol ChIP was performed usign the ChIP-IT High Seneitivity kit (Active Motif 53040). Chromatin shearing to 200-500 bp fragments was performed by sonication. 30 µg of sheared chromatin was subjected to immunoprecipitations using anti-FLAG M2 beads (Sigma-Aldrich M8823). Eluates were treated with Proteinase K.
Libraries were constructed using the TruSeq ChIP Library Preparation Kit (Illumina) according to the maufacturer's instructions.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
Data processing Raw data were quality checked using FastQC (v0.11.9). The average Phred score is 35.
Single-end reads were aligned to the human rDNA using default bowtie2 (v2.3.5.1) arguments.
Duplicated reads were marked with Picard (v3.0.0) using the following arguments: MarkDuplicates VALIDATION_STRINGENCY=LENIENT ASSUME_SORTED=true REMOVE_DUPLICATES=FALSE
Using samtools (v1.10), .bam files were filtered to remove duplicated, unmapped, not primary alignment, and low-quality reads with the following arguments: view -F 1804 -q 30
Bedgraph files were generated using bedtools (version 2.27.1) with the following arguments genomecov -ibam .bg -scale RPM. Bedgraph files were then converted to bigwig files using bedGraphToBigWig (v4)
Assembly: Human ribosomal DNA (accesion number U13369.1)
Supplementary files format and content: Aligned reads in bigwig format
Submission date Apr 26, 2023
Last update date Jan 30, 2024
Contact name Katherine Bohnsack
Organization name University Medical Centre Göttingen
Street address Humboldtallee 23
City Göttingen
ZIP/Postal code 37073
Country Germany
Platform ID GPL20301
Series (2)
GSE230433 GPATCH4 regulates rRNA and snRNA 2'-O-methylation in both DHX15-dependent and -independent manners
GSE230637 GPATCH4 regulates rRNA and snRNA 2’-O-methylation in both DHX15-dependent and -independent manners [ChIP-Seq]
BioSample SAMN34381871
SRA SRX20108495

Supplementary file Size Download File type/resource 70.2 Kb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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