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| Status |
Public on Jun 23, 2021 |
| Title |
4sUSeq_C1_Auxin_R1 |
| Sample type |
SRA |
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| Source name |
U2OS-SPT6-AID-C1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS-SPT6-AID-C1
treatment: Auxin
label: 15min 4sU
sirna: none
antibody: NA
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| Growth protocol |
U2OS-SPT6-AID-C1, U2OS-SPT6-AID-C2 and U2OS-WT cells were grown in DMEM supplemented with 10% FCS and 1% penicillin/streptomycin
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| Extracted molecule |
total RNA |
| Extraction protocol |
ChIP-seq: Cells were treated with 1% formaldehyde for 5 min at 37°C. After cell lysis, nuclei were re-suspended in RIPA buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP40, 1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA) and DNA was fragmented to a size <300 bp using Covaris. Chromatin was immunoprecipitated with 15 µg of corresponding antibody for 6 hours. Chromatin was eluted with 1% SDS and crosslinking was reverted overnight. For purification, chloroform/phenol extraction was used.
ChIP-seq: Libraries were prepared according to instructions accompanying the NEBNext Ultra™ II DNA Library Prep Kit for Illumina. Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (200 bp) and eluted. DNA fragments were amplified by 12 to 17 cycles of PCR and library size was tested with the Fragment Analyzer (Advanced Analytical). The amount of library DNA was quantified using a picogreen assay
RNA-Seq: U2OS cell lysates were spiked 4sU-treated mouse T-cell lysates. Total RNA was isolated using QIAzol and purified with miRNeasy columns and biotinylated. This was followed by 4sU-RNA pulldown pulldown using streptavidin-MyOne beads, followed by purification with RNA minElute columns (Qiagen)
RNA-Seq: 4sU-RNA was used for library preparation with NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and NEBNext Ultra II Directional RNA Library Prep kit. The libraries were amplified with 10-14 PCR cycles depending on the input RNA. The concentration and size distribution of the libraries were determined on a Fragment Analyzer using the NGS Fragment High Sensitivity Analysis Kit (1-6,000 bp; Agilent Technologies). The libraries were sequenced on the NextSeq500 Illumina platform for 75 cycles. Base calling was performed using Illumina’s BaseSpace platform.
3' polyA mRNA-Seq: RNA was isolated using RLT buffer and purified with RNeasy columns and biotinylated. After addition of ERCC spike in (mix1) (Thermo Fisher Scientific), alkylation of RNA was carried out using 10 mM iodoacetamide (Thermo Fisher Scientific), and the reaction quenched with 1M DTT (Thermo Fisher Scientific). Alkylated RNA was purified with RNA minElute columns (Qiagen).
3' polyA mRNA-Seq: Library preparation was carried out with using QuantSeq kit (Lexogen), and fragments were amplified with 13 PCR cycles.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
nascent RNA
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| Data processing |
Fastq files were generated Illumina’s BaseSpace platform and overall sequencing quality was analyzed with the FastQC script.
For ChIP-seq, fastq files were mapped to the human genome (hg19) using Bowtie v2.2.7. and normalized to the sample with the spike in control (mm10).
For 4sU-seq, reads were mapped to hg19 with Bowtie2, reads mapping to rRNA,exons and blacklist regions were removed, and then normalized based on read depth.
For SLAM-seq, reads were mapped to hg19 with Bowtie2. BAM files were normalized based on read depth.
Genome_build: hg19
Supplementary_files_format_and_content: bedGraph files as defined by UCSC (https://genome.ucsc.edu/goldenpath/help/bedgraph.html).
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| Submission date |
Nov 27, 2020 |
| Last update date |
Jun 23, 2021 |
| Contact name |
Elmar Wolf |
| E-mail(s) |
elmar.wolf@biozentrum.uni-wuerzburg.de
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| Organization name |
Biocenter of the University of Würzburg
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| Department |
Department of Molecular Biology and Biochemistry
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| Lab |
Cancer Systems Biology Lab
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| Street address |
Am Hubland
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| City |
Würzburg |
| State/province |
Bavaria |
| ZIP/Postal code |
97074 |
| Country |
Germany |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE162264 |
Targeted protein degradation reveals direct roles of SPT6 in POL2 elongation and termination |
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| Relations |
| BioSample |
SAMN16940738 |
| SRA |
SRX9598430 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4948108_4sUSeq_C1_Auxin_R1_minusStrand.bedGraph.gz |
60.1 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM4948108_4sUSeq_C1_Auxin_R1_plusStrand.bedGraph.gz |
57.5 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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