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| Status |
Public on Jun 18, 2022 |
| Title |
K562 ChRO-seq Olaparib Replicate B |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
treatment: 90 minutes Olaparib
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| Treatment protocol |
For the PARP1 inhibition experiment, cells were treated with either 10 μM Olaparib, initially diluted in DMSO, or DMSO only, for 90 min before immediately crosslinked for Micro-C or lysed in NUN buffer (0.3 M NaCl, 1 M Urea, 1% NP-40, 20 mM HEPES, pH 7.5, 7.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 20 units per ml SUPERase In Rnase Inhibitor (Life Technologies, AM2694), 1X Protease Inhibitor Cocktail (Roche, 11 873 580 001)) for ChRO-seq.
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| Growth protocol |
K562 cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1X penicillin streptomycin antibiotic.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For chromatin isolation from cultured K562 cells, we added 1 ml of 1X NUN buffer and vigorously vortexed the samples for 1 min. An additional 500 µl of NUN buffer was added and samples were vigorously vortexed for an additional 30 s. The samples were then incubated on ice for 30 min with a brief vortex every 10 min and centrifuged at 12,500g for 30 min at 4 °C, after which the NUN buffer was removed from the chromatin pellet. The chromatin pellet was washed 3 times with 1 ml of 50 mM Tris-HCl, pH 7.5, supplemented with 40 U/ml of RNase inhibitor, centrifuged at 10,000Xg for 5 min at 4 °C, and the supernatant discarded. After washing, 100 µl of chromatin storage buffer (50 mM Tris-HCl, pH 8.0, 25% glycerol, 5 mM Mg(CH3COO)2, 0.1 mM EDTA, 5 mM DTT, 40 U/ml Rnase inhibitor) was added to each sample. The samples were loaded into a Bioruptor and sonicated with the power setting on high, with a cycle time of 10 min with cycle durations of 30 s on and 30 s off. The sonication was repeated up to three times, as needed, to get the chromatin pellet into suspension. Samples were stored at −80 °C.
ChRO-seq library preparation was performed following a published protocol(18), with minor modifications. Chromatin from 1 million K562 cells in 25 µl chromatin storage buffer was mixed with 25 µl of 2X chromatin run-on buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2,1 mM DTT, 300 mM KCl, 10 μM Biotin-11-ATP (Perkin Elmer, NEL544001EA), 40 μM Biotin-11-CTP (Perkin Elmer, NEL542001EA), 10 μM Biotin-11-GTP (Perkin Elmer, NEL545001EA), 40 μM Biotin-11-UTP (Perkin Elmer, NEL543001EA), 2ng/μl Yeast tRNA (VWR, 80054-306), 0.8 U/μl RNase inhibitor, 1% Sarkosyl (Fisher Scientific, AC612075000)). The run-on reaction was incubated in a thermomixer at 37 °C for 5 min at 750 RPM. The reaction was stopped by adding 300 μl of Trizol LS (Life Technologies, 10296-010). To clean up the reaction prior to base hydrolysis, 40 μl of 1-bromo-3-chloropropane (BCP) (Sigma, B9673) were added to the samples and samples were vortexed for 20 sec, incubated for 3 min at RT and centrifuged at 17,000Xg at 4°C for 5 min. ~250 μl of aqueous phase was transferred to a new 1.5 ml tube and samples were pelleted at 17,000Xg at 4°C for 15 min in 650 ml ice-cold 100% EtOH with GlycoBlue (2.5 μl) (Ambion, AM9515) to visualize the RNA pellet. The pellet was further washed with 0.5 ml ice-cold 75% by vortex-mixing and centrifugation at 17,000Xg at 4°C for 5 min. Any residual EtOH was removed by air-drying the pellet for 5 min in RT. The RNA pellet was resuspended in 20 μl of diethylpyrocarbonate (DEPC)-treated water and heat denatured at 65 °C for 40 sec. For base hydrolysis, 5 μl of 1N were added to the RNA sample to get 0.2N NaOH, and samples were incubated on ice for 8 min. Base hydrolysis was stopped by adding 25 μl of Tris-HCl pH 6.8 and gently mixing. 3′ Adapter ligation was done using T4 RNA Ligase 1 (NEB, M0204L). A first binding to streptavidin beads (NEB, S1421S) and washed as described (18). RNA was removed from beads by Trizol (Life Technologies, 15596-026) and followed by a 5′ decapping with RNA 5′ pyrophosphohydrolase (RppH, NEB, M0356S). The 5′ end was phosphorylated using T4 polynucleotide kinase (NEB, M0201L). A second bead binding was performed, and an on-beads 5′ adapter ligation followed. After bead washes, RNA was removed from beads by adding 300 μl of TRI reagent (MRC, TR 118). 40 μl of BCP were added to the samples and samples were vortexed for 20 sec, incubated for 3 min at room temperature and centrifuged at 17,000Xg at 4°C for 5 min. ~180μl of aqueous phase was transferred to a new 1.5 ml tube and samples were pelleted at 17,000Xg at 4 °C for 15 min in 450 ml ice-cold 100% EtOH with GlycoBlue (2.5 μl) to visualize the RNA pellet. The pellet was further washed with 0.4 ml ice-cold 75% EtOH by vortex-mixing and centrifugation at 17,000Xg at 4 °C for 5 min. Any residual EtOH was removed by air-drying the pellet for 5 min in RT. A reverse transcription reaction using Superscript III Reverse Transcriptase (Life Technologies, 18080-044) was used to generate cDNA. cDNA was then amplified using Q5 High-Fidelity DNA Polymerase (NEB, M0491L) to generate the ChRO-seq libraries following the protocol provided by NEB.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChRO-seq
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| Data processing |
All data processing for ChRO-seq data (as well as PRO-seq and GRO-seq available raw data) in this study was done using the Proseq2.0 pipeline available from GitHub (https://github.com/Danko-Lab/proseq2.0).
Assembly: hg38
Supplementary files format and content: bigwig file - contains raw read counts
Library strategy: ChRO-seq
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| Submission date |
Jun 13, 2022 |
| Last update date |
Jul 21, 2022 |
| Contact name |
Charles Grahe Danko |
| E-mail(s) |
dankoc@gmail.com
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| Organization name |
Cornell University
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| Department |
Baker Institute for Animal Health
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| Street address |
235 Hungerford Hill Road
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| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE206025 |
RNA polymerase II and PARP1 shape enhancer-promoter contacts [ChRO-seq] |
| GSE206133 |
RNA polymerase II and PARP1 shape enhancer-promoter contacts |
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| Relations |
| BioSample |
SAMN29012113 |
| SRA |
SRX15693017 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6239052_K562_ChROseq_Olaparib_B_minus.bw |
36.0 Mb |
(ftp)(http) |
BW |
| GSM6239052_K562_ChROseq_Olaparib_B_plus.bw |
37.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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