title: GSM9467428: HEK293T GLO1 cells, Untreated_H3R17MGH1_1; Homo sapiens; OTHER
library_name: GSM9467428
library_construction_protocol: CUT&Tag was performed with HEK293T native nuclei (GLO1-/-) plus and minus MGO treatment (0.2 mM for GLO1-/-, overnight) using the EpiCypher CUTANA CUT&Tag Kit version 3 and following CUT&Tag protocol v3 with slight modifications. Briefly, for each reaction, 100K nuclei were immobilized to Concanavalin-A beads (ConA; EpiCypher, 21-1401) and incubated overnight (4 °C with gentle rocking) with 0.5 µg of primary antibodies listed in Supplementary Table 1. Bead-bound nuclei were collected on a magnetic rack and resuspended in 50 µL Wash Buffer 1 with 0.5 µL of anti-rabbit immunoglobulin G (IgG) secondary antibody and incubated RT for 30 min on a nutator. After washes in Wash Buffer 1, bead-bound nuclei were resuspended in 50 µL of Wash Buffer 2 with 2.5 µL of pAG-Tn5 (EpiCypher, 15-1017) for 1 hour incubation at RT on a nutator. Bead-bound nuclei were washed a total of two times with Wash Buffer 2, resuspended in 50 µL cold Tagmentation Buffer and incubated 1 hour in a thermocycler set to 37°C. After tagmentation, chromatin-bound fragments were released with 5 µL of the SDS Release Buffer during an 1 hour thermocycler incubation at 58°C and quenched with 15 µL SDS Quench Buffer. For Library amplification, 2 µL of i5 unique index primer (10 µM), 2 µL of i7 unique index primer (10 µM), and 25 µL of Non-Hot Start 2X PCR Master Mix was added to each reaction and was subjected to PCR (58°C for 5 min; 72°C for 5 min; 98 °C  for 45s and for 14 cycles 98°C for 15 s, and 60°C for 10 s; 72°C for 1 min). To purify libraries 1.3 x volume of SPRIselect reagent (Beckman Coulter, B23317) was added to each reaction and incubated for 5 min RT. Libraries were twice washed with 85% EtOH and eluted in 17 µL 0.1X TE Buffer.