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| Status |
Public on Dec 16, 2022 |
| Title |
HEK293 Ctrl ChIP-Seq |
| Sample type |
SRA |
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| Source name |
kidney
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| Organism |
Homo sapiens |
| Characteristics |
tissue: kidney
cell line: Human embryonic kidney 293 cells
chip antibody: IgG (Santa Cruz)
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| Treatment protocol |
Infection of HEK293 cells with lentiviruses carrying either shCr or shTHOC5. The treatment was applied for 4 days.
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| Growth protocol |
HEK293 cells were grown in Dulbecco´s modified Eagle's medium supplemented with 10% (v/v) FCS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5x10^6 cells were fixed in 1% PFA for 5 minutes and subsequently quenched in 125 mM glycine. Nuclear fraction was isolated and resuspended in sonication buffer (0.1% SDS, 50 mM TrisHCl, pH 8.0, 0.2 mM EDTA, 1x Protease inhibitor cocktail (Sigma)). Chromatin was sheared into 500 bp DNA fragments using Covaris AFA™ (Adaptive Focused Acoustics, Woburn, MA, USA) technology according to the manufacturer’s instructions. After shearing, NaCl and NP40 were added to final concentration of 150 mM and 1%, respectively. Aliquots of extracts were precipitated by pre-coated Protein G Agarose-PLUS (Santa Cruz Biotechnology Inc.) with anti- RNA polymerase II (Abcam), THOC5 (Bethyl Laboratories. Inc.), CDK12 (Cell signaling), FLAG M2 antibodies or control IgG. Following 4 h rotation at 4°C, the beads were washed three times in RIPA buffer (150 mM NaCl, 1% NP40, 0.1 % SDS, 50 mM TrisHCl at pH 8), and two times in wash buffer (500 mM NaCl, 1% NP40, 0.1 % SDS, 100 mM TrisHCl at pH 8). Cross-links were reversed for 8 h at 65°C (250 mM NaCl) in the presence of RNAse A. Following proteinase K digestion at 55°C for 1 hour, the bound DNA fraction was isolated using NucleoSpin Extract II (Macherey-Nagel, Dueren, Germany).
5 ng of ChIP derived DNA were subjected to library preparation using TruSeq ChIP Library Preparation Kit (Illumia). Libraries were prepared and indexed according to manufacturer’s protocol. Indexed libraries were pooled and sequenced on an Illumina HiSeq 2500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Nanopore: Basecalls performed using Guppy version 3.3.3+fa743a6
Nanopore: Mapping using minimap2
Nanopore: Trimming of long read sequencing data using NanoFilt version 2.5.0
ChIp-Seq: FASTQ files were generated by CASAVA (v1.8.2). Galaxy workflow for RNA-Seq (www.usegalaxy.org) was used for subsequent data analysis. Reads were mapped to the human reference genome (GRCh38) using Bowtie2 (Galaxy Version 2.3.4.1). Metagene analysis was performed using deepTools 2.0
TT-Seq: Raw reads were mapped to the human reference genome (GRCh38) using Bowtie2 (Galaxy Version 2.3.4.1). Read coverage of individual gene was normalized and visualized using Seqmonk. To measure the progression of Pol II molecules into the gene body we applied a pipeline described by Gregersen et al. (33) for calling RNA Pol II transcription wave peak positions and elongation rates from DRB/TT-seq time-series data using R. Metagene analysis was performed using deepTools 2.0.
Assembly: GRCh38
Supplementary files format and content: For the identification of 3’cleavage sites the tool NanoFilt (Filtering and trimming of long read sequencing data) was used to trim the mRNA sequences from 5’end to a uniform length so that we keep always the last 200 nucleotides (python get_read_ends.py --bases_from_end 200 reads.fastq.gz | gzip > last_200_bp.fastq.gz). Trimmed reads were aligned to the GRCh38 human genome reference using minimap2. The quantification of 3’ cleavage was perform with Seqmonk using the human polyA database as reference. Genes that contain more than two annotated polyadenylation sites (PAS) and more than 5 reads at distal PAS sites were selected. The ratios between proximal- and distal cleavage site were calculated.
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| Submission date |
Jun 21, 2022 |
| Last update date |
Dec 16, 2022 |
| Contact name |
Mareike Polenkowski |
| E-mail(s) |
[email protected]
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| Organization name |
Medical School Hannover
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| Department |
Cell Biochemistry
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| Lab |
Tamura-Niemann
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| Street address |
Carl-Neuberg-Straße 1
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| City |
Hanover |
| ZIP/Postal code |
30625 |
| Country |
Germany |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE173374 |
THOC5 complexes with DDX5, DDX17 and CDK12 to regulate R loop structures and transcription elongation rate |
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| Relations |
| BioSample |
SAMN29224673 |
| SRA |
SRX15815351 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6257279_IgG.bigwig |
207.1 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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