|
| 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 |
| E-mail(s) |
katherine.bohnsack@med.uni-goettingen.de
|
| 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] |
|
| Relations |
| BioSample |
SAMN34381871 |
| SRA |
SRX20108495 |
