ATACseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

The goal of this study is to analyse the open chromatin regions via ATACseq in different sub-types of the zebrafish embryonic dorsal aorta (DA) at 28-30 hpf using TgBAC(runx1P2:Citrine);Tg(kdrl:mCherry) double-transgenic zebrafish embryos. 2000-3000 cells were FAC-sorted directly into 100 µl of 1x HBSS/10 mM HEPES/0.25% BSA buffer for Tagmentation (as described by Buenrostro et al. (2013), Nat. Methods 10, 1213-1218), but with 1.5 µl Tn5 transposase (Illumina) in a 50 µl reaction volume. DNA was purified using the QIAquick PCR purification kit (Qiagen). Fragments were amplified for 16 cycles. Genomic control DNA was isolated from 30.000 cells using the DNeasy Blood & Tissue kit (Qiagen) and eluted with EB buffer. Tagmentation was performed as described. Fragments were amplified for 9 cycles. Sequencing was performed on a NextSeq machine. Sequenced reads were checked for base qualities, trimmed where 20% of the bases were below quality score 20, and filtered to exclude adapters using Trimmomatic (Version 0.32). Overall design: Analysis of 5 different cell types; DN (double negative), SP-kdrl (single positive), DP-R1lo (double positive runx1 low expression), DP-R1med (runx1 medium expressionand) and DP-R1hi (runx1 high expression) in TgBAC(runx1P2:Citrine);Tg(kdrl:mCherry) double-transgenic zebrafish embryos, in triplicates, NextSeq with a 75 bp paired end