GSM5941054: Shear Input Rep1; Homo sapiens; ChIP-Seq
GSM5941054
RNA for RNA-Seq was extracted with RNeasy Micro Kit (Qiagen). Cells were cross-linked with formaldehyde (final concentration 1 %) for 5 min, sonicated and the cromatin was used for H3K27ac ChIP. For ATAC HUVEC were first frozen in the medium containing 10 % DMSO and used for OMNI-ATAC after thawing. For zebrafish RNA-Seq 300 embryos per condition were collected in ice cold 10 % DMSO and kept at -80°C for 2 min. Afterwards, embryos were washed twice with 1x Hanks'Balanced Salt Solution (HBSS, Gibco, # 14185-052)and deyolked with calcium-free Ringer's solution. Embryos were then dissociated with trypsin-EDTA (0.5 %) and collagenase type IV (50 mg/ml). The reaction was stopped by addition of FCS (5 %) andcells were collected by centrifugation at 350 g for 5 min at 4°C and washed twice with 1x HBSS followed by centrifugation. After a final wash, cells were resuspended in 1x HBSS, passed through a 40 m nylon filter to obtain a single cell suspension, which was subjected to fluorescence-activated cell sorting (FACS). EGFP positive ECs were collected in RLT buffer (RNeasy Micro kit, Qiagen). RNA Seq libraries were prepared using TruSeq Stranded Total RNA Sample Prep with RiboZero depletion. ChIP-Seq libraries were prepared using TruSeq ChIP Sample Preparation Kit (Illumina). ATAC-Seq libraries were prepared using the protocol by Buenorosto et al (Buenorosto, Nat Met, 2013)