GSM5703394: K562_KRAB_NS_5d_nodox_H3K9me3; Homo sapiens; OTHER
CUT&RUN for the K562 KRAB and CHO-K1 KRAB cell lines was performed according to the third version of the published protocol on protocols.io. For each antibody condition, 250,000 K562 or CHO-K1 cells were harvested and incubated with 10uL of activated Concanavalin A beads. Cells were permeabilized with 0.05% digitonin and incubated at room temperature for two hours with a 1:100 dilution of one of the following antibodies: guinea pig anti-rabbit IgG H&L chain (Antibodies-Online; Cat. No.: ABIN101961); rabbit anti-H3ac (Active Motif; Cat. No.: 39139); rabbit anti-H3K4me3 (Active Motif; Cat. No.: 39159); rabbit anti-H3K9me3 (Abcam; Cat. No.: ab8898). Unbound antibody was washed out, and cells were incubated with 140ng/mL of a protein A and micrococcal nuclease fusion (pA-MNase; generously provided by the Henikoff lab). Unbound pA-MNase was washed out, and cells were resuspended in a low-salt buffer. Tubes were placed in a thermal block chilled to 0°C, and 10mM CaCl2 was added to trigger DNA cleavage by pA-MNase. After 5 minutes, the reaction was stopped by chelating calcium ions with the addition of 20mM EGTA, and tubes were incubated at 37°C for 30 minutes under physiological salt conditions to promote diffusion of fragmented chromatin into the supernatant. Fragmented chromatin in the supernatant was collected and purified using the DNA Clean & Concentrator-5 kit (Zymo; Cat. No.: D4004). CUT&RUN for the CHO-K1 HDAC4 cell lines was performed using the EpiCypher CUTANA ChIC/CUT&RUN Kit (Cat. No.: 14-1048) according to the User Manual Version 2.0 with the following conditions: an input of 500,000 CHO-K1 cells per antibody condition; a final digitonin concentration of 0.01%; an overnight antibody incubation at 4°C with 1:100 dilutions of the anti-H3Kac and anti-H3K9me3 antibodies described above along with a 1:50 dilution of rabbit anti-H3K27me3 (Cell Signaling Technologies, Cat. No: 9733S); and DNA purification with the provided spin columns. Dual-indexed sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs; Cat. No.: E7645L) with 12-14 cycles of PCR, and size selection was performed with either Agencourt AMPure XP (Beckman Coulter; Cat. No.: A63880) or SPRIselect (Beckman Coulter; Cat. No.: B23318) magnetic beads. Library concentrations were quantified with the Qubit 1X dsDNA HS Assay Kit (Invitrogen; Cat. No.: Q33231), and library fragment sizes were assessed with the High Sensitivity D1000 ScreenTape (Agilent; Cat. No.: 5067-5584) and High Sensitivity D1000 Reagent (Agilent; Cat. No.: 5067-5585) on an Agilent 4200 TapeStation System. Libraries were pooled for genome-wide sequencing.