title: GSM6704363: Neutrophils_Shig_flexneri_M90T_rep2_input; Danio rerio; ChIP-Seq
library_construction_protocol: Tg(lyz::dsRed)nz50 zebrafish larvae were dissociated using 0.5% Trypsin EDTA 10x no phenol (Fisher cat no.15400054) and incubated twice for 10 min at 28.5oC. The reaction was stopped by adding 10% fetal calf serum. The suspensions were sieved using 70 μm Nylon cell strainers (Merck CLS431751-50EA) and then gently centrifuged for 3 min at 800g. Cells were then washed with 1mL PBS with 10 mM HEPES and 1 mM EDTA. Cells were incubated with fixable APC Cy7 (1:2000, Thermofisher cat no. L34994) dye for 30 min at 4oC. cells were fixed with 1% formaldehyde for 8 min at RT with occasional mixing. To stop the reaction 100 µL of glycine 1.25 M were added and the samples were incubated for 5 min at RT. After centrifuging at 600g for 10 min at 4oC, cells were washed with ice cold PBS containing protease inhibitor cocktail (Sigma-aldrich cat no. 5892970001) and centrifuged again. Cells were then resuspended in PBS with 10mM HEPES and 1 mM EDTA and run through a Becton Dickinson FACSAria III cell sorter. Chromatin was prepared for ChIPseq using the True MicroChIP Kit (Diagenode cat no. C01010130) and was sheared using Bioruptor® Pico sonication device (Diagenode cat no. B01060001) combined with the Bioruptor® Water cooler for 5 cycles using a 30'' [ON] 30'' [OFF] settings. Libraries were prepared using IP-Star® Compact Automated System (Diagenode cat no. B03000002) from input and ChIP'd DNA using MicroPlex Library Preparation Kit v2 (12 indices) (Diagenode cat no. C05010013). Libraries were pooled and sequenced with Illumina Novaseq 6000, running NovaSeq Control Software 1.6.0, with paired-end reads of 50bp length.