Multiplex oligos

The in situ Hi-C protocol from the 4DN Nucleosome Consortium was followed. Briefly, five million cells were crosslinked for each experiment and DpnII was used for DNA digestion followed ligation. To shear the DNA after ligation, ultra-sonication (Sonic Dismembrator model 500, Fisher Scientific) was used with ten timed on-pulses of 5 s each. Subsequently, to generate the library, each end of the isolated DNA fragments (isolation performed using Dynabeads MyOne Streptavidin T1 beads) was trimmed and ligated with the following adaptor oligonucleotide duplexes: Hi-C1_for and Hi-C1_rev, Hi-C2_for and Hi-C2_rev, Hi-C3_for and Hi-C3_rev, Hi-C4_for and Hi-C4_rev, and then amplified using the oligonucleotides HiC_for and HiC_rev (Supplementary Table 1). Alternatively, the NEBNext Ultra II Kit (NEB E7645) and Multiplex Oligos (NEB E7600S) were used for making the library. Four independent experiments from three biological replicates were combined for analyses. As controls, we used in vitro stimulated wild-type B cells and control KO B cells as previously published^{8 (link)}.

The NEB Next rRNA depletion kit (E6310) was used for RNA purification, as several samples had RNA integrity numbers <8 (but >7). Most samples had RNA integrity numbers >8. After rRNA depletion, the NEBNext UltraII Directional Kit (E7765) and NEB Multiplex Oligos (Kits-E7335,E7710) were used to generate and barcode sample libraries. Library concentrations were quantified by quantitative polymerase chain reaction using KAPA Quant assays, and values were used to generate an equimolar pool to ensure equal distribution of reads across all samples.

Total RNA (10 μg) was treated with 20 U of DNase I (Promega) at 37 °C for 40 min and then purified by phenol/chloroform extraction. Total RNA (5 μg) was subjected to mRNA purification using oligo d(T)25 beads (NEB #E7490S). cDNA was synthesized from purified RNA (10–40 ng) using NEBNext RNA library prep master mix set for Illumina (NEB #E6110S) and then purified using AMPure XP beads (Beckman Coulter, A63880). cDNA libraries were prepared using NEBNext ultra DNA library prep kit for Illumina (NEB #E7370S) with multiplex oligos (NEB #7500S).
The cDNA samples were sequenced on the Illumina HiSeq 2000 platform to obtain 76-bp single-end reads. Sequenced reads were mapped to the fission yeast reference genome (version ASM294v2.30) using the STAR program (v2.5.2) (ref. 58 (link)). htseq-count software (v0.6.1) was used to estimate read numbers assigned to exons, and the read numbers were normalized using the TMM method^{59 (link),60 (link)}. Differentially expressed genes between wild-type and cut14-208 condensin mutant cells were identified with the edgeR package using the dispersion value of 0.029, which was estimated from the cut3-477 and cut14-208 condensin mutant samples treated as biological replicas^{61 (link)}. FDR < 0.05 was used as a threshold to define significantly up- or downregulated genes.