NextSeq 550 paired end sequencing; CUT&RUN of Sox2 binding in the mouse P0 visual thalamus (dLGN)
CUT&RUN of Sox2 binding in the mouse P0 visual thalamus (dLGN)
dLGNs were dissected suspended in 1.5 ml of cold CUT&RUN wash buffer (HEPES pH 7.5 [20 mM], NaCl [150 mM], Spermidine [0.5 mM], Roche Complete Protease Inhibitor EDTA-Free (Cat. # COEDTAFRO, Roche) and then manually dissociated by gentle pipetting. Cells were collected by centrifugation at 600g for 3 minutes and the supernatant carefully removed, and then washed 2 more times by resuspension in 1 ml wash buffer. 4 µl of ConA bead slurry (prepared in binding buffer as described), were added per pair of dLGNs and the cells and beads were incubated for 10 min on a rotator. While the rest of the dissections proceeded, bead bound cells were kept in storage buffer (wash buffer with EDTA [2 mM]). 3 brains (6 dLGNs) were collected per sample. Once all dLGNs were collected, the beads were collected on the magnet and resuspended in 150 µl antibody buffer (wash buffer with EDTA [2 mM] and 0.025 % digitonin). Antibodies were added at 1:100 dilution and incubation proceeded ON at 4 °C. Antibodies used included anti-SOX2 (ABIN2855074, antibodies online), anti-SOX2 (ABIN2855073, antibodies online), and anti-HA (05-902R, Merck). One SOX2 sample was with only ABIN2855074, the other was with both SOX2 antibodies together. The two SOX2 biological replicates were performed independently from different litters of mice. The next day samples were washed twice with dig-wash (wash buffer with 0.025% digitonin) and then resuspended in 150 µl dig-wash containing pA-MNase (New England Biolabs 700 ng/ml, received as a gift from Steven Henikoff) at 1:200 dilution and rotated for 1 hr. Samples were washed twice and then resuspended in 100 µl dig-wash and equilibrated in ice. 2 µl 100 mM CaCl2 was added, and digestion proceeded for 30 min in ice. 100 µl 2X STOP buffer (NaCl [340 mM], EDTA [20 mM], EGTA [4 mM], digitonin [0.05%], RNase A [100 µg/ml], glycogen [50 µl/ml]) was added to stop the digestion reaction and incubated at 37 °C for 30 min. Samples were pelleted at 16000 g for 5 min and placed on the magnet rack. The supernatant was harvested, and beads were discarded. DNA purification was performed with phenol chloroform. Library preparation was performed using the KAPA HyperPrep Kit (Cat. #KK8504, KAPA Biosystems) according to manufacturer's instructions, using KAPA DUI adapters at 0.15 µM.