GSM9753963: MOF-dTAG, dTAG-neg, LS, Rep2; Mus musculus; Hi-C
At 3.5 h of dTAG13 treatment, Hoechst 33342 was added to the cell media (1:1000) and the cells were incubated at 37°C for the next 30 minutes. Following 4 h of dTAG13/ dTAG-Neg (or DMSO) treatment, cells were detached from the plate by addition of TrypLE and subsequent incubation for 1-2 min at 37°C. The cells were then washed once in media (Neurobasal media + DMEM/F12 in 1:1 ratio) and crosslinked in 1% formaldehyde for 10 minutes at room temperature (RT), followed by quenching with 125 mM glycine for 5 minutes. Cells were then centrifuged at 300 x g for 5 minutes and washed with media. Cells were resuspended in media and using FACS were sorted into G0/G1, ES, LS and G2/M stages based on the relative Hoechst 3342 fluorescence intensity. Cells were then centrifuged at 300 x g for 5 minutes and washed with media. Cell pellets were resuspended in cold permeabilization buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal CA-630, 1x PIC) and incubated on ice for 15 minutes. Nuclei were extracted using NEXSON (peak power 75 W, duty factor 2%, 200 cycles/burst, 4 °C, 10-30 seconds). Nuclei were pelletted and swelling was performed by resuspending them into 50 μl of 0.5% SDS and incubated for 10 minutes at room temperature. SDS was quenched adding 25 μl of 10% Triton X-100, 145 μl of water and 25 μl of NEBbuffer r2.1. Digestion was performed using 400 units of HindIII (NEB, 20 U/μl) at 37 °C overnight with intermittent rotation. Nuclei were washed twice with 1x NEBuffer 2 to remove the restriction enzyme. Biotin end fill-in was performed in a reaction volume of 150 μl using biotin-14-dCTP, dATP, dGTP, and dTTP (50 mM each), with 30 U of Klenow fragment at 25 °C for 1 hour. Proximity ligation was conducted for 4 hours at room temperature on a rotator using T4 DNA ligase in a reaction volume of 1 ml (1x Ligase buffer, 0.8% Triton X-100, 0.1 μg/μl recombinant albumin, 4 U/μl T4 DNA ligase). After ligation, nuclei were pelletted and the excess of biotin was removed from the un-ligated ends by T4 DNA polymerase treatment for 30 min at 20 °C in a 100 μl reaction volume (25 μM dATP, 25 μM dGTP, 30 U T4 DNA polymerase, 1x NEBuffer r2.1). The reaction was stopped adding 20 mM final EDTA and nuclei were pelletted again. Protein-DNA crosslinks were reversed by overnight incubation with Proteinase K at 65 °C. DNA was precipitated and resuspended into 130 μl of EB. DNA was sonicated to a size of 300-500 bp using Covaris E220 sonicator (peak power 140 W, duty factor 10%, 200 cycles/burst, 4 °C, 90 seconds, into 130 μl tubes). DNA quality was checked on capillary electrophoresis (Fragment Analyzer). Biotinylated fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads as described into the original in situ HiC method. Up to 50 ng of DNA linked to streptavidin beads was used to generate sequencing libraries Libraries were constructed using the NEBNext Ultra II DNA library preparation kit for Illumina