GSM5115035: Tri-4C-GLN_J1; Mus musculus; OTHER
Tri-4C was performed with modifications from a pre-print protocol (BioRxiv doi: https://doi.org/10.1101/743005). Briefly, 1 x 107 crosslinked mESCs were resuspended in cold lysis buffer (10 mM Tris-HCl pH 8.0, 10 mM NaCl, 0.2% Igepal CA630 (Sigma)) with 1x protease inhibitors followed by incubation on ice for 15 minutes and centrifuge at 4°C. The pellet was then washed with cold lysis buffer and resuspended in 0.5% SDS (Sigma). After incubated at 62°C for 7 minutes, 10% Triton X-100 (Sigma) was added to obtain the nuclei. Subsequently, in situ digestion was done overnight with MboI (NEB), CviQI (NEB), and NlaIII (NEB) endonucleases. The digested chromatin was washed twice to remove remaining enzyme. End blunting was performed by adding dNTP (Invitrogen), DNA polymerase I Klenow (NEB) and T4 DNA polymerase (NEB) followed by incubation at 37°C for 1 hour with rotation. Blunt end ligation was then carried out by adding T4 ligase buffer (NEB), 10% Triton X-100 (Sigma), BSA (NEB) and T4 ligase (NEB) followed by rotating at room temperature for 4 hours. The chromatin was then resuspended in 1 x T4 ligase buffer (NEB) with Proteinase K and SDS and incubated at 65°C for reverse crosslinking. The resulting DNA was purified by phenol-chloroform extraction and sonicated with Covaris S220 at 140 watts for 80 seconds. The fragmented DNA was size-selected using 0.40x/1.0x of Ampure XP beads (Beckman Coulter) and eluted in 10mM Tris-HCl. Subsequently, the sonicated fragment ends were repaired and A-tailed followed by purification with 1.0x of Ampure XP beads (Beckman Coulter). The A-tailed DNA fragments were then ligated with pre-annealed Y-shape adaptor and purified twice with 1.0x of Ampure XP beads (Beckman Coulter). Size distribution of final preamplification libraries was determined by Fragment analyzer with the DNF 474 kit. To amplify the Tri-4C library targeting the designated anchor, nested PCR was conducted for 14 cycles in the first round and 14 cycles in the second round.