title: GSM9220308: Astrocytes Donor A73 R1 BG4; Homo sapiens; OTHER
library_construction_protocol: (CUT&Tag) Astrocyte cultures were crosslinked by adding 31.25 uL of 16% formaldehyde to 1 mL of cell suspension in PBS + 0.04% BSA to reach a final concentration of 0.5%. Samples were incubated on ice for 5 minutes and quenched with 50 uL of 2.5 M glycine. Cells were pelleted at 300 x g for 5 minutes and washed once with PBS. Cells were lysed in chilled lysis buffer [10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl_2, 0.1% Tween-20, 0.1% NP-40 substitute, 0.01% digitonin, 1% BSA, 1 mM DTT, and 1x protease inhibitors] using 200 uL per large sample or 100 uL for small samples. Lysis was performed on ice for 5 minutes, followed by two washes with 1 mL wash buffer [same composition as above without NP-40 substitute]. Nuclei were resuspended in PBS + 0.04% BSA and counted. 100,000 nuclei were aliquoted per CUT&Tag reaction. (CUT&Tag) Nuclei were resuspended in 70-90 uL of incubation buffer, antibodies, and pAG-Tn5 added. The following antibodies and enzymes were added per reaction: Primary antibody: G4 scFv (Millipore MABE917, 0.25 mg/mL), 1.03 uL; Secondary antibody: Anti-FLAG M2 (Millipore F1804, 1 mg/mL), 1.48 uL; pAG-Tn5 transposome (EpiCypher, Cat# 15-1025-FT002, 3.94 uM dimer), 2.53 uL. Samples were incubated overnight at 4 oC with gentle rotation. Tagmentation was triggered by adding 4 uL of 250 mM MgCl_2 and incubating at 37 oC for 1 hour at 550 rpm. The reaction was quenched with 20 uL of 80 mM EDTA. DNA was purified using the Zymo DNA Clean & Concentrator-5 kit and eluted in 20 uL of nuclease-free water. PCR reactions (50 uL) included 25 uL NEBNext High-Fidelity 2x Master Mix and 2.5 uL of 10 uM indexed i7/i5 primers. Thermal cycling was done at 72 oC for 5 min; 98 oC for 30 s; 15 cycles of 98 oC for 10 s, 63 oC for 30 s, 72 oC for 1 min; final extension at 72 oC for 2 min. SPRIselect beads (0.75x) were used for purification, and libraries were eluted in 10 uL of water. DNA concentration was measured by Qubit, and libraries were diluted to 1 ng/uL for TapeStation QC.