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| Status |
Public on Aug 23, 2018 |
| Title |
Streptavidin_pull-down_midS_rep2 |
| Sample type |
SRA |
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| Source name |
HeLa S3 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa S3
cell type: cervical carcinoma
ezh2i treatment: Non treated
cell cycle: Mid S (3 h and 15 min after release)
spike-in: NO
labelling: EdU
antibody: N/A
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| Treatment protocol |
For all ChOR-seq and ChIP-seq, HeLa S3 cells were synchronized at the G1/S border by single thymidine block (2 mM, 17 h) and released into fresh media containing deoxycytidine (24 μM) for the indicated amount of time (See Cell cycle information). Parental samples were collected 1 hour before labelling. DNA was labelled during 20 min with medium containing 5-ethynyl-2'-deoxyuridine (EdU, 10 μM) and Nascent chromatin was collected (T0). Then, the remaining cells were washed with PBS, resuspended into new medium containing deoxycytidine and incubated at 37ºC for the indicated amount of time. To avoid entering into a second round of replication, thymidine (2mM) was added to the medium after 10 h of release and cells were incubated until reaching 24 h post-thymidine release. For experiments using EZH2 inhibitor (EPZ-6438, 1 μM), inhibitor was added to the medium 30 min before collecting Nascent chromatin and kept in culture medium until the end of the experiment. For each time-course experiment, replicated DNA was isolated by single Streptavidin pull-down on Nascent chromatin. In ChOR-seq analysis, asynchronous Drosophila S2 chromatin was labelled with EdU (10 μM) for 39 h and was used as internal control. Harvested cells were immediately fixed in 1% formaldehyde for 15 min. Fixation was stopped by adding Glycine (0.125M) and incubating for 5 min at RT.
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| Growth protocol |
HeLa S3 were grown in suspension in spinners in DMEM medium (Thermo Fisher Scientific) supplemented with dialyzed FBS (Invitrogen) and penicillin and streptomycin at 37ºC and 5% CO2. Asynchronous Drosophila S2-DRSC cells were grown in suspension in spinners in M3+BPYE (Sigma-Aldrich) media with 10% HI-FBS and penicillin and streptomycin at 25ºC as described in Drosophila Genomics Resource Center.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lysed during 20 min in ice-cold lysis buffer (100mM NaCl, 66mM Tris HCl pH8, 5mM EDTA, 0.3% SDS, 1.6% Triton X-100) supplemented with proteases inhibitors, passed through a 21G needle and sonicated using a Bioruptor nextGen (Diagenode) to obtain chromatin frgament size of 250-500bp. Sonicated chromatin was centrifuged at 14000rpm at 4ºC for 10 min and the supernatant used for subsequent steps. In parallel, and independently, Drosophila S2 cells were fixed, lysed and sonicated as described for Hela S3 cells. After sonication, HeLa S3 input chromatin required for the analysis of all time-points from the same time course experiment was mixed with Drosophila S2 chromatin (2.5% of total chromatin). Chromatin associated to each histone mark was obtained by incubating sonicated chromatin with the corresponding antibody.
Libraries were costructed using the KAPA Hyperprep kit following manufacturer’s instruction, except that 1.25 µM of Illumina compatible indexed adapters (Pentabase) were ligated to A-tailed DNA. Following adapter ligation biotin-TEG-azide (Berry&Associates) was linked to EdU in Click reaction. Biotinylated products were captured using MyOne Streptavidin T1 beads (Thermo Fisher Scientific) and subjected to PCR amplification following the KAPA Hyperprep kit protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Illumina RTAv2 software was used for basecalling
Reads were aligned to human genome assembly (GRCh37/hg19) and D. melanogaster genome assembly (BDGP R5/dm3) by Bowtie v. 1.1.2 using parameters -m1 --best. Mapping and subsequent analysis of the data were done using local Galaxy server and custom R scripts
PCR duplicates were removed using RmDup v.2.0 and unique mapped reads were extended to 250 bp (H3K4me3) or 500bp (H3K27me3) to account for the average library size.
Reads were summed in 500 bp non-overlapping bins.
For ChIP-seq samples, reads were normalized to Reads Per Million (RPM).
For ChOR-seq samples, reads were normalized to Reference-adjusted RPM (RRPM) dividing human values by total Drosophila unique mapped reads.
Peak calling was performed with MACS v. 2.0.9 under broad domain parameters using Parental ChIP-seq of Total H3 from each experiment as a reference.
Genome_build: hg19 and dm3
Supplementary_files_format_and_content: bedgraph files were generated using Galaxy. Scores represent the sum of RPM or RRPM in 500bp windows
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| Submission date |
Feb 08, 2018 |
| Last update date |
Aug 23, 2018 |
| Contact name |
Anja Groth |
| E-mail(s) |
anja.groth@cpr.ku.dk
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| Organization name |
Novo Nordisk Foundation Center for Protein Research
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| Street address |
Blegdamsvej 3B
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| City |
Copenhagen |
| ZIP/Postal code |
2200 |
| Country |
Denmark |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE110352 |
Accurate recycling of parental histones reproduces the histone modification landscape during DNA replication |
| GSE110354 |
Accurate recycling of parental histones reproduces the histone modification landscape during DNA replication |
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| Relations |
| BioSample |
SAMN08499496 |
| SRA |
SRX3665693 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2988409_Replicated_regions_MidS_rep2.bed.gz |
465.1 Kb |
(ftp)(http) |
BED |
| GSM2988409_Streptavidin_pull-down_midS_RPM_rep2.bedgraph.gz |
32.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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