title: GSM4680722: LSEC_AT_GATA4_KO2; Mus musculus; ATAC-seq
library_construction_protocol: Livers were perfused in situ via the portal vein with a 0.05% collagenase/amino acid/saccharide solution, dissected, mechanically disrupted, pooled from three mice, digested at 38 °C in collagenase/Gey's balanced salt solution and filtered through a 250 µm mesh. Non-parenchymal cells were separated by a 19.3 % Nycodenz  gradient. Afterwards, LSEC were isolated by magnetic-activated cell sorting (MACS) using anti-CD146 MicroBeads according to the manufacturers' instructions (Miltenyi Biotech). Purity of LSEC isolates was confirmed by flow cytometry and showed at least 95 % positivity for CD31 and/or Stabilin-2. LSEC cells were isolated from three 1 year old control and GATA4 knockout mice, followed by ATAC-seq, using a modified protocol from (PMID:28846090). Briefly, 50.000 fresh-isolated LSEC were incubated in 50 μL cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Igepal CA-630) for 3 minutes. The lysis was stopped by adding with 1 ml resuspension buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) and the nuclei were pelleted by 10 minute centrifugation (500 x g, 4°C). Isolated nuclei were incubated in transposition reaction mix containing 2.5 µl Nextera Tagment DNA Enzyme TDE (Illumina #15027916) in 47.5 µl 2x transposition buffer (20 mM Tris-HCl pH 7.6, 10 mM MgCl2, 20% Dimethylformamide) for 30 min at 37 °C. Immediately following the transposition reaction, purification was carried out using ChIP DNA Clean and Concentrator Kit (Zymo #D5205). Sequencing libraries were performing using NEB Next High-Fidelity 2x PCR Master Mix (New England Biolabs #M0541S) and Nextera Index Kit (Illumina #15055290). Magnetic bead purification with two-sided size selection using undiluted Agencourt AMPure XP Beads (Beckman Coulter #A63881) ensured library sizes between 150 and 1000 bp. The quality of the libraries was analyzed by Bioanalyzer High Sensitivity DNA analysis kit (Agilent #5067-4626).
attributes.tag: GEO Accession
attributes.value: GSM4680722