A study of Dynamics of synthetic yeast chromosome evolution shaped by hierarchical chromatin organization. Sijie Zhou et al.
Description
1.We comprehensively sequenced this SCRaMbLEd pool and detected over 260,000 rearrangement events via a loxPsym junction analysis method.By analyzing each rearrangement event, we uncovered a stable rearrangement landscape for synthetic chromosomes. 2.To investigate the mechanisms underlying the rearrangement patterns,we compared the rearrangement map with local chromatin structures and the three-dimensional genome architecture via assay for transposase-accessible chromatin using sequencing (ATAC-seq) and genome-wide chromosome conformation capture (Hi-C), respectively
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ATAC-seq: Immediately following the nuclei prep, the pellet was resuspended in the Tn5 transposase reaction mix. The transposition reaction was carried out for 30 min at 37 °C. Tn5 transposed DNA were purified by AMPure DNA magnetic beads. A qPCR reaction was performed on a subset of the DNA to determine the optimum number of PCR cycles. The amplified libraries were run on an Agilent Tapestation 2100 (Agilent Technologies) to detect size distribution of library fragments. Biological replicates were performed in duplicate for all ATAC experiments. Hi-C: Restriction fragment ends were labeled with biotinylated cytosine nucleotides by biotin-14-dCTP (TriLINK) followed by ligation. Purified DNA was sheared to a length of ~400 bp. Point ligation junctions were pulled down with Dynabeads MyOne Streptavidin C1 (Thermofisher). The Hi-C library for Illumina sequencing was prepped by NEBNext Ultra II DNA library Prep Kit for Illumina (NEB) according to the manufacturers’ instructions. Fragments between 400 and 600 bp were paired-end sequenced on an illumina Nova-seq PE150 platform. Hi-C sequencing The genomic DNA from exponential phase cells was cross-linked and digested with 200U MboI (NEB) as previously described (Lieberman-Aiden et al.,2009). Restriction fragment ends were labeled with biotinylated cytosine nucleotides by biotin-14-dCTP (TriLINK) followed by ligation. Purified DNA was sheared to a length of ~400 bp. Point ligation junctions were pulled down with Dynabeads MyOne Streptavidin C1 (Thermofisher). The Hi-C library for Illumina sequencing was prepped by NEBNext Ultra II DNA library Prep Kit for Illumina (NEB Cat#E7645S) according to the manufacturers’ instructions. Fragments between 400 and 600 bp were paired-end sequenced on an Illumina Nova-seq PE150 platform.