biostudies-arrayexpressTina KaragyozovaHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14419Our previous work on the histone H3.3 chaperone HIRA revealed its importance for maintaining the targeting of H3.3 to its pre-existing sites. In concert with replication fork-coupled deposition of the replicative H3.1 variant, this establishes boundaries of H3.3/H3.1, which define early replication initiation zones, which are disrupted in the absence of HIRA (Gatto et al., 2022). We have also recently shown a role of HIRA for active gene organisation and compartment A interactions by Hi-C. Here, we performed a HIRA rescue experiment in HIRA KO HeLa cells combined with a G1/S synchronisation to assay the recovery of H3.1 and H3.3 distribution (prior to S phase entry) by SNAP capture-seq and the pattern of nascent DNA synthesis in early S phase (2h) by EdU-seq. We also include H3.3 SNAP capture-seq from an asynchronous HIRA rescue we performed to assay recovery of genome organisation by Hi-C (submitted as a separate ArrayExpress entry).biostudies-arrayexpressSample Collection - For H3.1/H3.3-SNAP ChIP-seq, we collected either G1/S-arrested cells by a double thymidine block (t0) or asynchronous cells (asynch). For EdU-seq, used to map sites of ongoing DNA synthesis, we released G1/S synchronized cells in S-phase as described in (Gatto et al., 2022). Ninety minutes after release of cells from the G1/S block, we performed a 30min pulse labelling by adding EdU (25μM) to the medium. For all experiments, we collected 4 million cells/condition.Nucleic Acid Extraction - We performed SNAP capture-seq of G1/S-synchronised cells by double thymidine block as described in (Forest et al., 2024; Gatto et al., 2022).Sequencing - Samples were sequenced on Illumina NovaSeq 6000 (PE100) at the Next Generation Sequencing (NGS) platform of Institut Curie.Growth Protocol - We used HeLa cells stably expressing H3.1-SNAP-HA or H3.3-SNAP-HA which were knocked out for HIRA (KO, CRISPR/Cas9 HIRA KO) and transfected them with plasmids expressing with YFP (control) or HIRA-YFP 48 prior to collection as in (Ray-Gallet et al., 2018). Cell lines were grown and synchronized at the G1/S transition using a double thymidine block as described in (Forest et al., 2024; Gatto et al., 2022). All cell lines were tested negative for mycoplasma.Library Construction - We prepared sequencing libraries at the Next Generation Sequencing (NGS) platform from Institut Curie with the Illumina TruSeq ChIP kit.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesMetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000ChIP-seqHomo sapiensHIRA defines early replication initiation zones independently of their genome compartmentTina KaragyozovaJean-Pierre QuivyAlberto GattoMarc Mart-RenomTina Karagyozova, Alberto Gatto, Audrey Forest, Jean-Pierre Quivy, Marc Marti-Renom, Leonid Mirny, Geneviève AlmouzniAudrey ForestLeonid MirnyGeneviève AlmouznifalseEdU-seq and H3 variant SNAP capture-seq in HIRA KO HeLa cells rescued with HIRAOur previous work on the histone H3.3 chaperone HIRA revealed its importance for maintaining the targeting of H3.3 to its pre-existing sites. In concert with replication fork-coupled deposition of the replicative H3.1 variant, this establishes boundaries of H3.3/H3.1, which define early replication initiation zones, which are disrupted in the absence of HIRA (Gatto et al., 2022). We have also recently shown a role of HIRA for active gene organisation and compartment A interactions by Hi-C. Here, we performed a HIRA rescue experiment in HIRA KO HeLa cells combined with a G1/S synchronisation to assay the recovery of H3.1 and H3.3 distribution (prior to S phase entry) by SNAP capture-seq and the pattern of nascent DNA synthesis in early S phase (2h) by EdU-seq. We also include H3.3 SNAP capture-seq from an asynchronous HIRA rescue we performed to assay recovery of genome organisation by Hi-C (submitted as a separate ArrayExpress entry).2025-08-07T00:00:00Z2025-08-08T00:01:52.837Z2024-09-04T21:29:34.599ZE-MTAB-14419ERP163839E-MTAB-14416E-MTAB-14415E-MTAB-14417EFO_0002944EFO_0004170EFO_0003789EFO_0002692EFO_0005518EFO_000418410.1101/2024.08.29.610220