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| Status |
Public on Jun 26, 2023 |
| Title |
U2OS_ChIPseq_Dyskerin_control_rep2 |
| Sample type |
SRA |
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| Source name |
U2OS
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| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
cell type: Human bone osteosarcoma
treatment: Vehicle
chip antibody: dyskerin (Abnova, H00001736-m04)
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| Treatment protocol |
250nM flavopiridol for 2 hours prior to cell harvest, added directly to culture medium.
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| Growth protocol |
DMEM, 10% fetal bovine serum, 1x penicillin/streptomycin. Cells were cultured at 37°C at 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
6 to 10 million cells were crosslinked for 5 minutes at room temperature with 16% methanol-free formaldehyde (Thermo Fisher Scientific) used at a final concentration of 1%. After quenching the reaction with 125 mM glycine final concentration, cells were washed twice in ice-cold PBS and collected by scraping. Pellets were resuspended in buffer A (5 mM PIPES pH 8.0, 85 mM KCl, 0.5% NP-40 substitute) and incubated on ice for 10 minutes. After centrifugation (5000 rpm for 5 minutes at 4°C), the supernatants containing the cytoplasm were removed and the nuclei-containing pellets were washed in buffer A without detergent. Pellets were then resuspended in shearing buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 0.1% SDS) and sonicated with a ME220 focused-ultrasonicator (Covaris). After clarification by centrifugation (13000 rpm for 10 minutes at 4°C), the supernatant was adjusted in composition to RIPA buffer and transferred to a new tube to be used for immunoprecipitation. Equal amounts of chromatin (as measured by DNA concentration) were incubated rotating overnight at 4°C with pre-conjugated beads (20 μl of Dynabeads and 3 μg antibody, dyskerin (Abnova, H00001736-m04) or RNAPII (MBL, MAB0601)) in RIPA buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 200 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS). Immunoprecipitates were washed once with each of the following: RIPA buffer, RIPA buffer high salt (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS), LiCl buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 250 mM LCiCl, 0.5% NP-40 substitute, 0.5% sodium deoxycholate), and twice with TE buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The beads were then resuspended in TE buffer with 0.5% SDS supplemented with proteinase K (New England Biolabs) and RNase A (Thermo Fisher Scientific) and incubated with shaking at 65°C for 4 hours. The DNA was recovered by phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation using standard procedures, and resuspended in TE buffer.
Libraries were generated using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer’s instructions.
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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 |
Sequencing quality checks were performed on all experiments using FastQC (v0.11.9). X
Adapter sequences were removed using TrimGalore (v0.6.4_dev) and reads shorter than 20 nt were discarded.
Reads were aligned to the human reference genome (GRCh38/hg38) using bwa-mem (v0.7.17-r1188) with default options.
Reads that failed to align and those with MAPQ ≤ 30 were filtered out, and PCR duplicates were discarded using the Picard MarkDuplicates (v2.18.11) module.
Genome coverage tracks in bigWig format were generated using the bamCoverage module from deeptools (v3.2.1) with --binSize 50 --normalizeUsing CPM options.
Assembly: GRCh38/hg38
Supplementary files format and content: bigWig, genomic coverage CPM-normalized
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| Submission date |
Aug 14, 2022 |
| Last update date |
Jun 26, 2023 |
| Contact name |
Federico Agostini |
| E-mail(s) |
[email protected]
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| Organization name |
Science for Life Laboratories
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| Street address |
Tomtebodavägen 23a
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| City |
Solna |
| State/province |
Stockholm |
| ZIP/Postal code |
171 65 |
| Country |
Sweden |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE211199 |
Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency [ChIP-seq] |
| GSE211202 |
Co-transcriptional pseudouridylation of mRNAs by the H/ACA complex controls translational efficiency. |
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| Relations |
| BioSample |
SAMN30308426 |
| SRA |
SRX17062405 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6456046_U2OS_ChIPseq_Dyskerin_control_rep2_mapq30_CPM.bw |
100.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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