GSM6336571: hNP-Mock; Homo sapiens; OTHER
neuronal cultures were dissociated using Accutase and 5 million cells were resuspended in 5 mL 10% FBS in PBS. 5 mL of 4% formaldehyde in 10% FBS in PBS was added and cells were crosslinked for 10 minutes. Glycine was added to a final concentration of 0.125M and cells were centrifuged at 300 rcf for 5 minutes. Cells were washed twice with PBS, then snap frozen and stored at -80 C. To amplify 4C libraries, PCR amplification was performed with 16 x 50 μL PCR reactions using Roche Expand Long Template polymerase (Roche 11759060001). Reaction conditions are as follows: 11.2 μL Roche Expand Long Template Polymerase, 80 μL of 10 X Roche Buffer 1, 16 μL of 10 mM dNTPs (Promega PAU1515), 112 μL of 10 M forward primer, 112 μL of 10 M reverse primer, 200 ng 4C template DNA, and H2O until 800 μL total. Reactions were mixed and then distributed into 16 x 50 μL reactions for amplification. Cycling conditions were: 2 minutes at 94 C, 10 seconds at 94 C, 1 minute at 63 C, 30 seconds at 68 C, repeat steps 2-4 but decrease annealing temperature by one degree, until 53 C is reached at which point the reaction is cycled an additional 15 times at 53 C, after 25 total cycles are performed the reaction is held for 5 minutes at 68 C and then 4 C. Libraries were cleaned-up using a Roche High Pure PCR purification kit (Roche 11732676001) using 4 columns per library. Purified 4C-seq libraries were resuspended in 120 μL of 10 mM Tris-HCl pH 7.5.