2.3. Hi-C, Illumina RNA-Seq, and PacBio Sequencing

Hi-C libraries was constructed according to a protocol on protocol.io [25]. Briefly, approximately 10⁷ cells were collected and fixed in formaldehyde, from which nuclei were isolated. Endonuclease (Dpn II) was then added and the nucleus sample was incubated at 62 °C for 20 min to digest DNA, followed by the incubation with a mixture of Klenow fragment, biotin-labelled dCTP, dTTP, dATP, and dGTP. After the ligation of fragmented DNA, proteinase K was added and the solution was incubated for 30 min. Then, the remaining DNA was precipitated and the biotin-labeled fragments were selected for library construction. Two Hi-C libraries were constructed and sequenced using the BGISEQ-500 platform. A total of 260 Gb raw data was produced.

To achieve a cost-effective deep sequencing of the transcriptome, an equal aliquot of RNA from each of the nutrient conditions (nutrient-replete, –Cu, –Mn, –Ni, + 1/5 Zn, and + 1/5 Fe) were combined to produce a RNA mixture > 5 μg, which was subjected to Illumina RNA-Seq sequencing (10 Gbp) and PacBio single-molecule sequencing (15 Gbp) using HiSeq × ten platform and PacBio ISO-Seq platform (BGI Genomics Co., Ltd.), respectively. All raw sequencing reads were deposited in the SRA database under Accession No. SAMN13258281. Illumina RNA-Seq sequencing raw reads were subject to quality filtering by removing sequences that contain adapters, >10% unknown nucleotides, or >50% of low quality (Q-value ≤ 10) bases. PacBio single-molecule sequencing reads were filtered to obtain high-quality full-length transcripts according to single molecular real-time (SMRT) link v5.0.1 pipeline provided by Pacific Bioscience [26]. Then, we used Illumina RNA-Seq reads to further correct the PacBio single-molecule reads by using Proovread software [27].