GSM8147008: Evf2TSTS Nono rep1 CutandRun; Mus musculus; OTHER
The protocol is described in detail (Cajigas et al. 2021, modified from Skene et al 2018). For Cut&Run, E13.5 ganglionic eminences were dissected from Evf2+/+ and Evf2TS/TS embryos in L15 medium (six embryos per genotype). Tissues were pooled for each genotype, triturated by pipetting and filtered through a cell-strainer capped 5 ml polystyrene roundbottomed tube (BD Falcon) to generate single-cell suspensions. Cells were counted using the Luna Automated Cell Counter (Logos Biosystems). The appropriate volume of cells to obtain 250,000 cells per sample was centrifuged at 600 g for 3 min at 4°C. The supernatant was removed and the cell pellet was gently resuspended in ice-cold wash buffer [20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5 mM spermidine and EDTA-free protease inhibitor cocktail]. The cells were centrifuged at 600 g for 3 min at 4°C. The supernatant was removed and cells were gently resuspended in 1 ml of wash buffer. Anti-Smarcc2 (Bethyl, Cat# A301-038A; RRID:AB_817992) and anti-Nono (Bethyl, Cat#A300-587A; RRID:AB_495510) were used for Cutand Run. CUT&RUN libraries were prepared using the KAPA Hyper Prep Kit protocol, with some modifications. The total volume of CUT&RUN DNA was used for library construction. For adapter ligation, 5 μl of 3 μM adapter stock from the KAPA Dual-Indexed Adapter Kit was used. The ligation was incubated at 20°C for 15 min. The library was amplified using the following cycling conditions: 14 cycles of 98°C for 45 s, 98°C for 15 s and 60°C for 10 s; 72°C for 1 min. After library amplification, the libraries were purified using 50 μl of KAPA Pure Beads and eluted in 20 μl of water. CUT&RUN sample quality was analyzed by TapeStation prior to sequencing on a NovaSeq 6000 (SP 100 cycles).