GSM8153281: LB2 pA-DamID, clone SC9, parental, replicate 2, dam only; Homo sapiens; OTHER
LMNB2 pA-DamID maps were generated as previously described (van Shaik, 2022, 36050765). Briefly, one million of HAP1 were collected by centrifugation (500 g, 3 min) and washed sequentially in ice-cold PBS and digitonin wash buffer (D-Wash) (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 0.02% digitonin, cOmplete Protease Inhibitor Cocktail). Cells were rotated for 2 hours at 4 °C in 200 μL D-Wash with 1:200 LMNB2 (Abcam, ab8983) followed by a wash step with D-Wash. Following this step, cells were incubated with a solution of D-Wash buffer and Rabbit-anti-mouse IgG (1:200, Abcam, ab6709), followed by a wash step. This incubation was repeated with a 1:200 pA-Dam solution (equivalent to nearly 60 Dam units from NEB), followed by two wash steps. Dam activity was induced by incubation for 30 min at 37 °C in 100 μL D-Wash supplemented with the methyl donor SAM (30 µM) while gently shaking (500 rpm). Genomic DNA was isolated and processed similarly to DamID, except that the DpnII digestion was omitted, and 65-bp reads were sequenced. For every condition, another 1 million cells were processed in only D-Wash and, during Dam activation, incubated with 4 units of Dam enzyme (NEB, M0222L). We use Dam control samples to account for DNA accessibility and amplification biases. Genomic DNA was isolated (Bioline, bio-52067) and ~ 500 ng were digested with DpnI (10 U, NEB, R0176L) in CutSmart Buffer 1X (8h 37°C, 20 min 80°C) in a total volume of 10 µL. A-tailing was performed by addition of 5 µL of the A-tailing mix (0.5 µL of Cutsmart buffer 10X, 0.25 µL Klenow 50 U/µL (NEB, M0212M), 0.05 µL dATP 100 mM, 4.2 µL H2O) and incubation 30 min at 37°C followed by 20 min at 75°C. Adapters were ligated by adding 15 µL of the ligation mix (3 µL T4 Ligase Buffer 10X, 0.5 µL T4 ligase (5 U/µL , Roche, 10799009001), 0.25 µl of x-Gene Stubby Adapter 50 mM (IDT), 11.25 µl H2O) and incubating samples 16h at 16°C and 10 min at 65°C. Finally, the Methyl Indexed PCR was performed by mixing 4 µL of ligated DNA with x-Gen Dual combinatorial Indexes (IDT) (125 nM final concentration) and MyTaq RedMix (Bioline, BIO-25048) in a final volume of 40 µL. The following PCR program was used: 1 X (1 min 94°C), 14 X (30 sec 94°C, 30 sec 58°C, 30 sec 72°C), 1X (2 min 72°C). Libraries were sequenced on Illumina platform Hiseq2500 with single end 65 bp reads.