characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2023-08-24T16:25:00Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2023-08-24T16:25:00Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA110130963
characteristics.characteristic: External Id
characteristics.text: Unit of Animal Genomics, GIGA Institute, University of Liege
characteristics.characteristic: INSDC center name
characteristics.text: 2023-08-24T16:25:00Z
characteristics.characteristic: INSDC first public
characteristics.text: 2023-08-24T16:25:00Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS12229784
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-11825:ATAC35_BOV1_f_Skin_snap_tn20_lib1
characteristics.characteristic: Submitter Id
characteristics.text: 2
characteristics.unit: month
characteristics.characteristic: age
characteristics.text: ArrayExpress
characteristics.characteristic: broker name
characteristics.characteristic: cell_type
characteristics.text: cattle
characteristics.characteristic: common name
characteristics.text: Protocols: Blood samples were collected from the tail or jugular vein using K2-EDTA blood collection tubes. White blood cells (WBC) were enriched by lysing erythrocytes using eBioscience 10X RBC Lysis Buffer (Thermo Fisher Scientific, Waltham, MA). Peripheral blood mononuclear cells (PBMC) were prepared by a density gradient centrifugation using either Ficoll-Paque PLUS (Cytiva, Marlborough, MA) or Lymphoprep (STEMCELL technologies, Vancouver, Canada) and SepMate-50 tubes (STEMCELL technologies). Animals were humanely euthanized and tissues were collected in ice-cold BELZER UW cold storage solution (Bridge to Life, London, United Kingdom) and processed in three ways. (1) Fresh sample (its file name contains a word fresh): Tissue kept in the UW cold storage solution was directly used for constructing ATAC-seq libraries in the day of sampling. (2) Cryopreserved sample (its file name contains slow): Tissue was cut into ~3 mm cubes and transferred to cryotubes filled with 1 ml of STEM-CELLBANKER DMSO-free cell freezing media (Amsbio, Abingdon, United Kingdom). The tubes were kept on ice for ~10 minutes and transferred in dry-ice during other samples being processed. The samples were stored in a -80 Celsius freezer until a library construction. (3) Snap-frozen sample (file name contains a word snap): Tissue was cut into ~3 mm cubes and transferred to cryotubes. The sample was snap-frozen in liquid nitrogen and stored at -80 Celsius until use. ATAC-seq libraries were constructed following the Omni ATAC-seq protocol (Corces et al., 2017) with some modifications. Tissue homogenization: A cryopreserved tissue in a vial was quickly thaw in a 37 Celsius water bath and transferred to an excess amount of ice-cold DPBS. The cryopreserved or fresh tissue was dissociated into a single-cell suspension using gentleMACS dissociator (with one or two cycles of the program B1. Miltenyi Biotec, Gladbach, Germany) with 3 ml of ice-cold Omni 1 x Homogenization Buffer in a gentleMACS C-tube. A snap-frozen sample was pulverized using a mortar and pestle chilled in liquid nitrogen. The cell suspension or pulverized tissue was transferred to a Dounce Tissue Grinder (D9063. Merck, Darmstadt, Germany) on ice with 3 ml of ice-cold Omni 1 x Homogenization buffer. The sample was homogenized with an A-pestle until resistance went away and further with a B-pestle (3 ~ 10 strokes each) so as to disrupt cellular plasma membranes. Cell debris was removed by passing the sample through stackable cell strainers (100, 70 and 30 um MACS SmartStrainers, Miltenyi Biotec). The flowthrough was further clarified by a brief centrifugation at 100 x g for 1 minute at 4 Celsius. Supernatant was mixed with an equal volume of ice-cold Omni 50% Iodixanol solution (final 25% Iodixanol). Purification of nuclei: Two layers of Iodixanol cushions were prepared in a 2 ml LoBind tube (Eppendorf, Hamburg, Germany) by placing 600 ul of ice-cold 40% Iodixanol solution on the bottom and overlaying 600 ul of ice-cold 29% Iodixanol solution using wide-bore tips. On the top, 800 ul of the cell suspension (25% Iodixanol) was placed. Density gradient centrifugation was done using a swing rotor at 6,000 x g for 30 minutes at 4 Celsius with a soft brake setting. Top layers were carefully removed down to ~2 mm above of the bottom layer. A nuclear fraction between the bottom and middle layers was collected (~ 400 ul) to a new LoBind tube on ice. The number of nuclei was counted by mixing 20 ul of the sample with 20 ul of 100 x diluted Hoechst 33342 (Fisher) using a hemocytometer under a fluorescence microscope. Tagmentation: Approximately 50,000 nuclei each were transferred to two of 1.5 ml LoBind tubes filled with1 ml of ice-cold ATAC-RSB + 0.1% Tween-20 buffer and centrifuged at 500 x g for 10 minutes at 4 Celsius. After carefully removing supernatant, nuclei were resuspended in 50 ul of Omni-ATAC reaction mix containing Tn5 Transposase TDE1 enzyme (Illumina, San Diego, CA). As effectiveness of Tn5 enzyme varied slightly among tissue types, we used two different amounts of the enzyme per sample. After mixing the sample by pipetting six times using a P200 fine tip, tagmentation reaction was carried out using Eppendorf Thermomixer at 37 Celsius at 500 rpm for 30 minutes. The reaction was stopped by adding 300 ul of PB buffer in MinElute PCR purification kit (Qiagen, Venlo, Netherlands) and 10 ul of 3M sodium acetate, pH 5.2. The sample was mixed, kept at room temperature for 10 min and stored at -20 Celsius until DNA purification. Library preparation: The tagmented DNA was purified and eluted in 21 ul elution buffer using the MinElute PCR purification kit. The DNA was amplified using NEBNext High-Fidelity 2X PCR Master Mix with Ad1 and Ad2 primers (Buenrostro et al., 2013) for 13 PCR-cycles. The amplified libraries were purified and eluted in 51 ul elution buffer using the MinElute PCR purification kit. Library size distribution was monitored using 10 ul of the library by QIAxcel capillary electrophoresis (Qiagen). Large DNA fragments were eliminated using AMPure XP magnetic beads (Beckman Coulter, Brea, CA) by a right-side size selection using 0.55 x followed by 1.5 x ratio of beads to sample. Library concentration was estimated using KAPA Library Quantification kit (Kapa Biosystems, Wilmington, MA).
characteristics.characteristic: description
characteristics.text: infant
characteristics.characteristic: developmental stage
characteristics.text: normal
characteristics.characteristic: disease
characteristics.text: wild type genotype
characteristics.characteristic: genotype
characteristics.text: BOV1
characteristics.characteristic: individual
characteristics.text: not applicable
characteristics.characteristic: isolate
characteristics.text: Bos taurus
characteristics.characteristic: organism
characteristics.text: Bos taurus
characteristics.characteristic: organism
characteristics.text: skin
characteristics.characteristic: organism part
characteristics.characteristic: sampling site
characteristics.text: Bos taurus
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.characteristic: sex
characteristics.characteristic: strain
characteristics.text: snap-frozen tissue, TDE1=2.0 ul
characteristics.characteristic: tissue with known storage state
characteristics.text: ATAC35_BOV1_f_Skin_snap_tn20_lib1
characteristics.characteristic: title