title: GSM8860881: neural progenitor cells, C2, Pan troglodytes, IgG, NPC_Rep2; Pan troglodytes; OTHER
library_name: GSM8860881
library_construction_protocol: NPCs at D14 and piNs at D28 were scraped off of tissue culture plates into NE1 lysis buffer (20mM HEPES pH 7.9, 10mM KCl, 0.1% Triton X-100, 3mM MgCl2, 0.5mM Spermidine, 1 tablet Complete Protease Inhibitor EDTA-free per 50ml) and nutated at 4°C for 10min to isolate nuclei. Nuclei were pelleted at 4°C at 200rpm for 10min and then resuspended in 1ml WB150 (20mM HEPES pH 7.5, 150mM NaCl, 0.2% Tween-20, 1g/L BSA, 0.5mM spermidine, 10mM sodium butyrate, 1 tablet Complete Protease Inhibitor EDTA-free per 50ml) for counting. For each library, 500,000 nuclei were aliquoted into a total volume of 1ml WB150. 20µl Concanavalin A bead (Bangs Laboratories, cat # BP531) slurry was prepared per library by washing beads twice in binding buffer (20µM HEPES-KOH pH7.9, 10µM KCl, 1µM CaCl2, 1µM MnCl2) and resuspending in 10µl binding buffer. Beads were added to the nuclei suspension and incubated for 10min at room temperature. Using a magnet, the supernatant was removed, and beads were resuspended in 50µl antibody buffer (WB150 supplemented with 0.1% Triton X-100 and 2mM EDTA) with 1µl primary antibody (rabbit anti-FOSL2: Cell Signaling Technology, cat #19967; rabbit anti-MAZ: Abcam, cat #ab85725; rabbit IgG: Cell Signaling Technology, cat #2729S; rabbit anti-H3K27Ac: abcam, cat #ab4729; rabbit anti-FOS: Cedarlane, cat# 226003) for overnight incubation at 4°C. The next day, the primary antibody solution was removed and beads were resuspended in 100µl of antibody buffer with 1.2µl of secondary antibody (goat anti-rabbit: Fisher Scientific, cat #NBP172763; goat anti-mouse: Abcam, cat #ab46540). Beads were incubated at room temperature for 1h and then washed 3X with 1ml WB150. Beads were resuspended in 100µl WB300 (20mM HEPES pH 7.5, 300mM NaCl, 0.2% Tween-20, 1g/L BSA, 0.5mM spermidine, 10mM sodium butyrate, 1 tablet Complete Protease Inhibitor EDTA-free per 50mL) containing 2.5µl proteinAG-Tn5 (Epicypher Inc, cat #15-1017) and incubated at room temperature for 1h. Then, beads were washed 3X with 1ml WB300 and incubated in tagmentation buffer (WB300 supplemented with 10mM MgCl2) at 50°C for 1h at 500rpm. DNA was extracted with Phenol/Chloroform, ethanol precipitated with 0.02mg/ml GlycoBlue (Life Technologies, cat #AM9515), and resuspended in 22µl H2O. For library preparation, 20µl of DNA was PCR amplified with 5X KAPA Hifi Buffer (Roche, cat #KK2101), 0.3mM dNTP (Roche, cat #KK2101), 1µl KAPA HiFi Polymerase (Roche, cat #KK2101), and primers with Illumina adapters in a 50µl reaction at 72°C for 5min, 98°C for 30s, 14 cycles of 98°C for 10s, 63°C for 30s, 72°C for 1min, and 72°C for 5min. DNA was purified using double-sided size selection (0.5X, then 1.2X) with Ampure XP beads (Thermo Fisher, cat #NC0110018). Briefly, 25µl of SPRI beads were added to the PCR reaction, incubated at room temperature for 8 min, and allowed to adhere to the magnet for 8 min. The supernatant was moved to a new tube and 65µl of SPRI beads were added. After 8 min incubation at room temperature, beads were allowed to adhere to the magnet for 8 min, the supernatant was discarded, and beads were washed twice with 200µl 80% EtOH for 30s each. Beads were resuspended in 53µl 10mM Tris-HCl, pH 7.5 and placed on the magnet, and the supernatant was moved to a new tube. The supernatant was then further cleaned by adding 55µl beads, incubated at room temperature for 8 min, washed twice with 200µl 80% EtOH for 30s each, and then eluted in 20µl 10mM Tris-HCl, pH 7.5. Libraries were quantified with the KAPA Library Quantification Kit (Roche, cat #07960140001).