Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: PAIRED
Construction protocol: 75,000-100,000 acutely isolated cells from mouse, macaque, and human forebrain (or cultured 3T3 and HEK293T cells) were used for each experiment. Cells were resuspended in 500 mL ice cold Nuclei Isolation Buffer (NIB) (10 mM HEPES-KOH, Ph 7.9. 10 mM KCl, 0.1% NP40, 0.5 mM Spermidine and 1X Halt protease inhibitor cocktail) and incubated for 10 min in ice. Nuclear pellet was collected by centrifuging at 600xg for 3 min at 40C and again washed with 500 mL NIB. Nuclear pellet was collected by centrifugation at 600xg for 3 and resuspended in 100 mL of NIB. Successful nuclei isolation was confirmed by visual inspection under a phase contrast microscope. Next, 10 mL Concanavalin A lectin beads (Bio-Mag Plus) washed and resuspended in Binding Buffer (BiB) (20 mM HEPES-KOH, pH 7.9, 10 mM KCl, 1 mM CaCl2 and 1 mM MnCl2) were added to each sample and rotated for 10 min at room temperature. Nuclei bound Concanavalin A beads were pulled by placing the tubes on a magnetic stand to remove the supernatant. For LaminB GO-CaRT, anti LaminB1 antibody (Abcam# ab16048) was used and for SON GO-CaRT, anti-SON antibody (Atlas antibodies #HPA031755) was used at 1:100 dilution in Blocking Buffer (BlocB) (Wash buffer containing 2mM EDTA). Same steps were followed for histone marks using anti-H3K9me2 (Active Motif #39239, 1:100), anti-H3K9me3 (Abcam# ab8898; 1:100), anti-H3K27me3 (Cell signaling technologies #9733; 1:100). An IgG control was run in parallel for all experiments (Cell signaling technologies #2729; 1:100). To each sample 50 mL of BlocB containing primary antibody was added and incubated overnight on a rotator at 40C. Next day, samples were briefly spun and placed on magnetic stand to clear off the liquid. Samples were washed twice with 1mL Wash Buffer (WaB) (20 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.1% BSA, 0.5 mM Spermidine and 1X Halt protease inhibitor cocktail). At the end of two washing steps, Concanavalin A bound nuclei were resuspended in 50 mL of WaB and gently mixed. To each tube, 2.5 mL of pA-MNase (diluted 1:10 in WaB from 140 mg/mL stock) kindly provided by Steven Henikoff (Fred Hutchison Cancer Research Center) was added and rotated for 1 hour at 40C. Samples were briefly spun and placed on magnetic stand to remove the liquid. Samples were washed twice with 1mL WaB. At the end of two washing steps, Concanavalin A bound nuclei were resuspended in 100 mL of WaB and gently mixed. The tubes were placed in pre-chilled metal blocks sitting in ice. While gently vortexing, 2 mL of 100 mM CaCl2 was added to initiate MNase digestion and returned to pre-chilled metal blocks. Digestion was carried out for 30 min and stopped by adding 100 mL of 2XSTOP (200 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 mg/mL RNAseA, 40 mg/mL Glycogen and 10 pg/mL of heterologous DNA) while gently vortexing. To release pA-MNase cleaved fragments, samples were incubated at 370C for 15 min. DNA was extracted using Phenol: Chloroform extraction method. We considered the possibility that the pA-MNase cleaved DNA-protein complexes might be too large to diffuse out of the nucleus and enter the “soluble” fraction. To test this, we recovered DNA from both soluble and total fractions (soluble + insoluble). LaminB profiles were highly similar (Pearson= 0.94) between soluble and total fractions, indicating that DNA-protein complexes released by LaminB GO-CaRT can readily diffuse out of the nucleus. Sequencing libraries were prepared from GO-CaRT DNA fragments using KAPA HyperPlus with amplification DNA library preparation kit (#KK8512) following manufacturer instructions. The library was amplified for 13-15 PCR cycles and larger DNA fragments were depleted by doing a 0.55X size selection using Agencourt AMPure XP beads (Beckman #A63881).