GSM6938986: pLHiC-MYCN-Input-OHT; Homo sapiens; Hi-C
For phosphorylated linker Hi-C (pLHi-C), phosphorylated linker HiChIP (pLHiChIP) and spike-in phosphorylated linker HiChIP (spLHiChIP), cells were cross-linked with 1% formaldehyde for 5 min and stopped by adding 125 mM glycine for 10 min at room temperature. Nuclei were isolated and a one-step in situ digestion and dephosphorylation took place by using DpnII and rSAP enzymes. During the nuclei isolation step, only for the spLHiChIP samples, murine NHO2A cells were added as exogenous control (spike-in) at a 1:20 cell ratio. Next, biotinylated oligos with 5' end phosphate were used with T4 ligase for the ligation step. Chromatin fragmentation was performed using the Covaris Focused Ultrasonicator M220 for 30 min per ml lysate. Samples were de-crosslinked overnight and Protein and RNA were digested with proteinase K and RNase A. DNA was isolated using ChIP DNA Clean & Concentrator (Zymo Research). Biotin pull-down was performed with MyOne Streptavidin C1 beads (Thermo Fisher Scientific) according to manufacturer's instructions. Only for pLHiChIP and spLHiChIP samples, the chromatin fragmentation was followed by an immunoprecipitation of the target protein. To this end, Dynabeads Protein A and Protein G were pre-incubated overnight with 10-15 µg antibody in the presence of 5 mg/ml BSA. Next, chromatin was added to the pre-incubated beads for at least 6h at 4°C with rotation. Beads were then washed with different TRIS buffers containing distinct salt-concentrations, EDTA and detergents, followed by wahsing with only TE-buffer. The immunoprecipitated chromatin of the pLHiChIP and spLHiChIP samples was eluted with 100mM NaHCO3 and 1% SDS for 15 min. Eluted samples were de-crosslinked overnight. DNA was isolated using ChIP DNA Clean & Concentrator (Zymo Research). Biotin pull-down was performed with MyOne Streptavidin C1 beads (Thermo Fisher Scientific) according to manufacturer's instructions. Library preparation was the same for all samples and was done on beads using the NEBNext ChIP-Sequencing Library Prep Master Mix Set for Illumina (NEB) according to manufacturer's instrcutions. The only deviation from the instructions was an additional washing step between each module using a Tris-buffer with 0.5 mM EDTA, 1 M NaCl and 0.05% Tween followed by a washing step with only TE buffer. Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in single-read or paired-end mode on a NextSeq500 or NextSeq2000 platform (Illumina). pLHi-C sequencing (Illumina NextSeq500) and (s)pLHiChIP sequencing (Illumina NextSeq500 or NextSeq2000)