title: MLL2 facilitates long-range gene regulation through LINE1 elements [Micro-C]
design: MLL2 facilitates long-range gene regulation through LINE1 elements [Micro-C]
library_construction_protocol: mESC were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM (Thermo Fisher Scientific, 10829018) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, A5256801), 1x antibiotic and antimycotic solution (Sigma-Aldrich, A5955), 1x glutaMAX (Thermo Fisher Scientific, 35050038), 1x non-essential amino acids (NEAA; Thermo Fisher Scientific, 11140035), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, 21985023) and leukemia inhibitory factor (LIF; made in-house). Cells were maintained at 37 °C with 5% CO2. For the multilineage differentiation, cells were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific, 25030024), 0.1 nM β-mercaptoethanol, 1x NEAA, 1x antibiotic and antimycotic solution and 1 µM retinoic acid (RA; Sigma-Aldrich, R2625) for 4 days. mESC were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM (Thermo Fisher Scientific, 10829018) supplemented with 15% fetal bovine serum (FBS; Thermo Fisher Scientific, A5256801), 1x antibiotic and antimycotic solution (Sigma-Aldrich, A5955), 1x glutaMAX (Thermo Fisher Scientific, 35050038), 1x non-essential amino acids (NEAA; Thermo Fisher Scientific, 11140035), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, 21985023) and leukemia inhibitory factor (LIF; made in-house). Cells were maintained at 37 °C with 5% CO2. For the multilineage differentiation, cells were grown on 0.1% gelatin-coated plates, using KnockOutTM DMEM supplemented with 10% FBS, 2 mM L-glutamine (Thermo Fisher Scientific, 25030024), 0.1 nM β-mercaptoethanol, 1x NEAA, 1x antibiotic and antimycotic solution and 1 µM retinoic acid (RA; Sigma-Aldrich, R2625) for 4 days. Starting from a previously described Cre-inducible Mll2-KO mESC line, Mll1-KO mESC were generated via CRISPR editing. Two sg-RNAs were designed flanking the region to-be deleted for each line, using Benchling's CRISPR tool (https://www.benchling.com/crispr). For each sgRNA, two oligonucleotides were synthesized , annealed and cloned into a CRISPR-Cas9 expression vector (pX330-hCas9-long-chimeric-grna-g2p, provided by Leo Kurian's lab). mESC were transfected with CRISPR-Cas9 constructs using LipofectamineTM 3000 (Thermo Fisher Scientific, L3000001). For the generation of the double-KO mESC line, cells were treated with (Z)-4-Hydroxytamoxifen (4-OHT; Sigma-Aldrich, H7904) at a final concentration of 800 nM for 48 hours. The micro-C library was prepared using the Dovetail® Micro-C Kit according to the manufacturer's protocol. Briefly, the chromatin was fixed with disuccinimidyl glutarate (DSG) and formaldehyde in the nucleus. The cross-linked chromatin was then digested in situ with micrococcal nuclease (MNase). Following digestion, the cells were lysed with SDS to extract the chromatin fragments, and the chromatin fragments were bound to Chromatin Capture Beads. Next, the chromatin ends were repaired and ligated to a biotinylated bridge adapter followed by proximity ligation of adapter-containing ends. After proximity ligation, the crosslinks were reversed, the associated proteins were degraded and the DNA was purified. The purified DNA was converted into a sequencing library using Illumina-compatible adaptors. Biotin-containing fragments were isolated using streptavidin beads prior to PCR amplification. For each cell type (e.g. WT Day 0), micro-C samples were prepared as two biological replicates, and for each replicate three libraries were prepared.