NextSeq 500 sequencing; A gene regulatory network for neural induction
A gene regulatory network for neural induction
In brief, tissues were dissected in ice cold 1x PBS and transferred to low binding PCR tubes containing 50-100µL of 1x Protease Inhibitor Cocktail in 1xPBS (cOmplete mini EDTA-free; Roche) on ice. Where necessary, 0.12% trypsin or 1XPBS + 200mM EDTA was used to aid dissection. Treated tissues were rinsed with PBS to remove residual trypsin. Tissues were collected in batches every 30mins before tubes were spun at 100G/3min/4°C to gently pellet the tissue. Tubes were snap frozen in liquid nitrogen before storage at -80°C for up to 6 months. For pre-streak EGK XII-XIII embryos, the hypoblast was removed and central epiblast was collected from 6 embryos in triplicate. After removal of the underlying mesoderm and endoderm, the neural plate at HH6-7 was harvested from 20 embryos in triplicate. For 0, 5, 9 and 12h node induced and contralateral uninduced area opaca, 14-30 pieces of tissue were collected in triplicate. A further 60 pieces of 9h and 12h uninduced tissue were collected and processed separately after it was discovered that 30 pieces of uninduced tissue at these time points did not generate enough material for ChIP. Samples were spun at 3500 RPM and 4°C for 3mins and the supernatant was divided into 3 separate 0.5mL low binding PCR tubes: 200µL each into H3K27ac and H3K27me3 tubes and the rest into the input tube (≈60µL). To the experimental tubes, 2µL of H3K27ac (Active Motif 39133, lot# 31814008) or H3K27me3 (Active Motif 39155, lot# 31814017) antibody was added, and these were incubated overnight at 4°C. To the input, 80µL of low EDTA TE buffer was added and stored overnight at 4°C.  The following day, ChIP reaction tubes were spun briefly at 4°C to collect the solution. “IP” reaction beads were removed from the rotator and mixed to an even suspension by pipetting with a wide bore tip. Keeping the beads well mixed, 50µL of bead slurry was added to each immunoprecipitation and the tubes returned to the 4°C rotator for 3-4h to pull down ChIPed chromatin. Next, samples were washed according to Section G of the Active Motif Low Cell ChIP-seq manual and eluted with 100µL of Elution Buffer AM4. Libraries were prepared using the Next Gen DNA Library kit (Active Motif 53216) and Next Gen Indexing kit (Active Motif 53264) but using ProNex® size-selective beads (Promega) to enrich for mono and di-nucleosomes (≈200-500bp). DNAs were size-selected using 1.2x/0.4x ratios of ProNex beads to purify ≈200-500bp fragments.