NextSeq 500 paired end sequencing; A gene regulatory network for neural induction
A gene regulatory network for neural induction
Hensen's node was grafted into the area opaca of HH4 chick embryos and the epiblast in contact with the node was collected after 9 and 12 hours (9 and 12hrs induced); the contralateral area opaca from the same embryos was collected after 0, 9 and 12 hours (uninduced area opaca). Once sufficient tissues were collected, they were thawed on ice and the nuclei isolated in 2mL of 1x Homogenisation Buffer Unstable (HBU) with a Dounce homogenizer on ice. HBU 1x contains 5mM CaCl2, 3mM Mg(Ac)2, 10mM Tris pH7.8, 320mM Sucrose, 0.1mM EDTA, 0.1% NP-40, 0.02mM PMSF and 0.3mM ß-mercaptoethanol, dissolved in H2O. Tissues were ground using 15 strokes with the loose pestle and 20 strokes with the tight pestle. Homogenate was then passed through a 30µm nylon mesh filter into a fresh 2mL low binding microfuge tube. Next, nuclei were gently pelleted by centrifugation for 10mins/500g/4°C and the supernatant removed. The pellet was resuspended in 200µL of 1xHBU by gentle pipetting. Nuclei were counted by taking 6x10µL homogenate samples, staining them with DAPI and viewing on a haemocytometer. After counting, nuclei were washed with 1mL of ATAC-RSB+0.1% Tween-20 and spun for 10mins/500g/4°C to pellet. The supernatant was carefully removed and the nuclei pellet was resuspended in 50µL of transposition mix by pipetting. The volume of Tn5 transposase (Illumina, 20034197) added to each reaction was scaled to 0.1µL per 1000 nuclei, so that ATAC samples with different numbers of nuclei were treated equivalently.  Libraries were prepared exactly as described in (Corces et al., 2017), using the adapter sequences listed in (Buenrostro et al., 2013).